![6
RNA that has been isolated from the tissue of choice is first
separated by size via denaturing agarose gel electrophoresis.
Following electrophoresis, RNA is transferred from the gel to a
membrane by capillary action, after which it is cross-linked to the
membrane for immobilization, and hybridized with a labeled probe
for transcript detection. Northern blot analysis can be performed
using either radiolabeled or non-isotopically labeled DNA as
hybridization probes. The choice of label is generally dependent
on a number of considerations, including sensitivity and resolu-
tion. Radioactive DNA probes can be quickly prepared, offer rapid
detection, and, importantly, provide much higher sensitivity com-
pared to nonradioactive probes. For example, nucleotides labeled
with 32
P enable high specific activity probes up to 2×109
dpm/μg,
which can detect as little as 10 fg of target RNA, and it is widely
appreciated that sensitivity can be of paramount importance when
investigating novel transcripts and potential splicing variants whose
cellular abundance is not yet known. However, a drawback of the
radioactive method is the need for licensing as well as the safety
issues, both of which may limit the feasibility of radioisotope use in
the laboratory. Further, half-lives of some radioisotopes (i.e., 32
P)
are quite short, so reagents must be used quickly or purchased on
a regular basis. In contrast, the nonradioactive system utilizing
digoxigenin (DIG) is an alternative method for detection of cellu-
lar RNAs [1, 2] and offers unique advantages such as enhanced
probe stability and shorter film exposure times for detection.
In this chapter we describe methods for northern blot analysis
that utilize both radioactive and nonradioactive probe preparation.
However, the initial steps of northern blot analysis, including gel
electrophoresis transfer, and immobilization of RNA to membrane
are independent of the type of probe used [3].
1. Prepare gel casting tray by sealing ends of gel chamber with
tape or proper casting system. Place appropriate number of
combs in gel tray.
2. Prepare 1 % (w/v) agarose in a glass flask, add water, and heat
solution in a microwave until agarose is completely dissolved.
For 120 ml gel volume, dissolve 1.2 g agarose in 86.4 ml water.
3. Cool agarose solution to 65–70 °C in a water bath. Swirl occa-
sionally to prevent uneven cooling.
4. After cooling, add 21.6 ml formaldehyde and 12 ml of 10×
MOPS to a final volume of 120 ml.
5. Add 12 μl of SYBR green II RNA gel stain to the gel mix.
Alternatively, ethidium bromide can be added to the cooled
agarose solution (final concentration 0.5 μg/ml) or directly to
the RNA samples (see step 13).
6. Ensure that there is enough space between the bottom of the
comb and the gel tray (0.5–1.0 mm) to allow for proper well
formation and to avoid sample leakage (see Note 1).
3.1 Denaturing
Agarose Gel
Electrophoresis
Marc R. Reboll et al.](https://image.slidesharecdn.com/2464969-250609220113-c1bac12a/85/Rna-Mapping-Methods-And-Protocols-1st-Edition-M-Lucrecia-Alvarez-21-320.jpg)
![10
6. Add the template to a supplied ready-to-use reaction tube and
carefully mix the components by gently flicking tube with
finger.
7. Add 5 μl of [α-32
P] dCTP and carefully mix by pipetting up
and down.
8. Incubate reaction at 37 °C for 10 min.
9. Stop the reaction by adding 5 μl of 0.2 M EDTA.
10. Purify the labeled probe using Illustra MicroSpin G-25
Columns (GE Healthcare, Pittsburg, PA).
11. Resuspend the resin in the supplied column by vortexing.
12. Detach the bottom closure by turning the cap.
13. Spin the column for 1 min at 735×g.
14. Place the column into a 1.5 ml tube.
15. Add labeling reaction to the center of the resin surface without
disturbing the resin bed.
16. Spin for 2 min at 735×g to collect the sample at the bottom of
the tube.
17. Store purified probe at −20 °C or use immediately.
1. Mix 0.58 g NaCl, 1 g dextran sulfate and 9.5 ml sterile water
in 50 ml plastic tub for 10 min. Heat to 60 °C until solution
turns clear, then add 0.5 ml 20 % SDS. Mix gently to avoid
bubbles and incubate at 60 °C during next two steps.
2. Denaturate 1 % yeast RNA and 10 mg/ml herring sperm DNA
by boiling in water bath for 5 min, then chilling on ice for
5 min.
3. Clean a hybridization tube, preheat to 60 °C, and insert blot
into pre-warmed tube.
4. Add 0.5 ml herring sperm DNA and 0.5 ml RNA to the
preheated hybridization buffer; mix and carefully pipet into
the hybridization tube. Avoid bubbles!
5. Incubate the tube for prehybridization at 60 °C for a
minimum of 30 min.
6. Radioactive probe can either be added directly to the tube or
mixed with a new aliquot of hybridization buffer, which
replaces the prehybridization buffer.
7. Incubate at 60 °C overnight or a minimum of 8 h.
1. Following hybridization, carefully remove the radioactive
hybridization buffer by pipetting or draining from tube. Buffer
can be directly disposed into radioactive waste or stored at
−20 °C for a maximum of 1 week and used once more for
hybridization (see Note 4).
3.3.2 Prehybridization
and Hybridization
with a Labeled DNA-Probe
3.3.3 Washing
Marc R. Reboll et al.](https://image.slidesharecdn.com/2464969-250609220113-c1bac12a/85/Rna-Mapping-Methods-And-Protocols-1st-Edition-M-Lucrecia-Alvarez-25-320.jpg)
![11
2. Wash the blot in the tube with Buffer A for 2 min to remove
residual, unbound radioactive probe.
3. Place blot in a glass dish and wash once again with Buffer A for
5 min with gentle agitation. Check counts using a hand coun-
ter and use this initial count as a baseline value of 100 % to
estimate the efficiency of next wash steps.
4. Wash blot in Buffer B at room temperature for 15 min with
gentle rocking. Check counts every 5 min. If the counts are
reduced to 3 %, skip the next step.
5. Wash in Buffer C for 15 min at 62 °C. Check counts every
5 min. If the counts are lower than 10 %, stop the wash.
6. Wash in Buffer D for 5 min at room temperature.
1. Wrap blot in plastic wrap and expose to X-ray film.
Typically, several hybridization experiments using different
labeled genomic probes are required to characterize all transcripts
from a single gene and provide visualization of an appropriate
positive control. Therefore, a single blot is usually probed several
times. However, prior to rehybridizing with new probes, the old
label should be carefully removed from the blot. This procedure
is called stripping.
1. First incubate the membrane in 0.1 % SDS at 95–100 °C for
10–20 min.
2. Next, wash the membrane in 2× SSC for 5 min at room tem-
perature and start the next round of hybridization with a new
probe. Expose blot to X-ray film to ensure that radioactive
probe is completely removed.
3. Begin new hybridization.
The digoxigenin (DIG) system—based on the steroid hapten,
digoxigenin, which occurs in certain digitalis plants—is an effective
method for nonradioactive labeling and detection of nucleic acids [4].
Digoxigenin is suitable for detection purposes based on three char-
acteristics. First, high affinity antibodies can be easily generated if
digoxigenin is coupled to a suitable carrier molecule. Second, there
are no endogenous background problems with the anti-digoxigenin
antibodies as in the case of other haptens, such as biotin, because
digoxigenin occurs exclusively in digitalis plants. Third, digoxigenin
can be coupled to nucleotides like dUTP and then incorporated
into nucleic acids using Klenow, Taq, or RNA polymerases to
generate DIG-labeled probes. These probes can be used in stan-
dard blotting and hybridization procedures and detected with
anti-digoxigenin conjugates such as fluorescent or alkaline-phos-
phatase labeled antibodies [5].
3.3.4 Detection
3.3.5 Stripping
and Reprobing
3.4 Nonradioactive
DNA-Probe Labeling
and Detection
RNA Full-Length Characterization](https://image.slidesharecdn.com/2464969-250609220113-c1bac12a/85/Rna-Mapping-Methods-And-Protocols-1st-Edition-M-Lucrecia-Alvarez-26-320.jpg)
![12
The nonradioactive DNA probe is labeled incorporating DIG-
labeled dUTP by PCR using the PCR DIG Probe Synthesis kit and
Anti-DIG-AP antibody for detection (see Subheading 2). This
method is particularly recommended when template is available in
limited quantity, is only partially purified, or is very short [6, 7].
The PCR DIG Probe Synthesis kit is especially designed for gen-
eration of highly sensitive hybridization probes suitable for detec-
tion of low (single) copy sequences.
1. In a 1.5 ml tube, prepare a DIG-labeled DNA probe that is
going to be used as hybridization probe adding the reagents in
the amounts shown in Table 1. Note that for DIG-labeled
probes <1 kb long, a 1:3 DIG-dUTP–dTTP ratio (standard ratio)
is recommended. However, for longer DIG-labeled probes
(i.e., >1 kb long), a 1:6 DIG-dUTP–dTTP ratio should be
used to avoid low yield of DIG-labeled PCR product.
2. In another 1.5 ml tube, prepare the unlabeled positive control
adding the reagents and amounts according to Table 1. Mix
the reagents and centrifuge briefly.
3. Transfer samples to 0.2 or 0.5 ml tubes suitable for PCR and
mix thoroughly.
4. Place samples in thermocycler and begin cycling program.
The optimal cycling conditions depend on the combination of
template, primers, and thermocycler (see Note 5). The follow-
ing conditions are a good starting point:
3.4.1 Probe Generation
Table 1
DIG-probe labeling by PCR using PCR DIG Probe Synthesis kit
Reagent
DIG-labeled
probe
Un-labeled
controla
Final concentration
Sterile double-distilled water Variable
volume
Variable
volume
–
10× PCR buffer with MgCl2 (vial 3) 5 μl 3 μl 1×
10× PCR DIG mix (vial 2) 5 μl – 200 μM
10× dNTP stock solution (vial 4) b
3 μl 200 μM
Forward and reverse primers [50 μM] 0.5 μl 0.3 μl 0.5 μM each primer
Enzyme mix (Expand High
Fidelity, vial 1)
0.75 μl 0.45 μl 2.6 units total enzyme
Template DNA Variable
volume
Variable
volume
10 ng genomic DNA
or 10 pg plasmid DNA
Final volume 50 μl 30 μla
30 or 50 μl
a
The use of a lower total reaction volume for the un-labeled control than the DIG-labeled probe is only to save reagents
of the PCR DIG Probe Synthesis kit (Roche Applied Science, Indianapolis, IN)
b
For DIG-labeled probes<1 kb long: use 1:3 DIG-dUTP–dTTP ratio and do not add 10× dNTP stock solution (vial 4).
For DIG-labeled probes>1 kb long use 1:6 DIG-dUTP–dTTP ratio and add 5 μl of 10× dNTP stock solution (vial 4)
Marc R. Reboll et al.](https://image.slidesharecdn.com/2464969-250609220113-c1bac12a/85/Rna-Mapping-Methods-And-Protocols-1st-Edition-M-Lucrecia-Alvarez-27-320.jpg)
![13
(a) Initial denaturation step (before the first cycle): 95 °C
for 2 min
(b) PCR amplification (35 cycles): 95 °C for 30 s; 60 °C
for 30 s; 72 °C for 40 s
(c) Final elongation step: 72 °C for 7 min
The labeling efficiency for PCR-labeled probes can be quickly
estimated by gel electrophoresis as described below:
5. Prepare a 1–1.5 % w/v agarose gel in TAE buffer and add 10 μl
SYBR®
Safe DNA Gel Stain 10,000× concentrate (Invitrogen,
Carlsbad, CA) per ml of agarose solution. Alternatively, gels
can be stained with ethidium bromide, as described above.
6. Load 5 μl of each PCR amplification product on agarose gel.
Include a DNA molecular weight marker. Electrophorese the
samples at 60 V for 40 min or until the DIG-labeled and unla-
beled probes are adequately separated. DIG-labeled probes are
larger than unlabeled probes; thus, these probes migrate more
slowly compared to unlabeled DNA of the same size. The inten-
sity of the stained DIG-labeled probe should be equal to, or
slightly less than, the intensity of the unlabeled probe DNA. If
these conditions are met, then the DIG-probe was likely labeled
efficiently and the standard amount of labeled probe (2 μl of
PCR product per ml hybridization buffer) can be used in hybrid-
ization reactions. If the intensity of the labeled PCR product
band is very strong on the gel, 0.5 μl probe per hybridization
buffer should be used (see Note 6). If the signal is very faint, up
to 4 μl probe per ml hybridization buffer should be used [5].
1. After fixing the RNA to a Zeta Probe membrane, or any similar
positively charged nylon membrane, incubate the membrane
in 2× SSC buffer to remove any agarose or salts remaining
from the RNA capillary transfer. First incubate the membrane
in 0.1 % SDS at 95–100 °C for 10–20 min.
2. Place membrane on 3MM paper with RNA face up.
3. For prehybridization and hybridization, use ready-to-use DIG
Easy Hyb solution. In a hybridization oven at 50 °C, pre-warm
a 50 ml tube containing 16 ml of DIG Easy Hyb solution.
4. Pre-warm an empty hybridization tube in the hybridization
oven at 50 °C.
5. Prehybridization step: insert membrane into the pre-warmed
hybridization tube and immediately add 10 ml of the
pre-warmed DIG Easy Hyb solution prepared in step 3
(see Notes 7 and 8). Incubate with rotation in a hybridization
oven at 50 °C for at least 30 min.
6. In a 1.5 ml tube, mix 200 μl Easy Hyb solution with the DIG-
labeled DNA probe (2 μl DNA probe labeled by PCR per ml
of DIG Easy Hyb solution) (see Note 6).
3.4.2 Prehybridization
and Hybridization
with a DIG-Labeled
DNA-Probe
RNA Full-Length Characterization](https://image.slidesharecdn.com/2464969-250609220113-c1bac12a/85/Rna-Mapping-Methods-And-Protocols-1st-Edition-M-Lucrecia-Alvarez-28-320.jpg)
![14
7. Denature probe by boiling 10 min at 100 °C, then immediately
quenching on ice.
8. Add the denatured probe to the remaining 6 ml of the pre-
warmed DIG Easy Hyb solution prepared in step 3 to obtain
6 ml of hybridization solution. If a previous hybridization solu-
tion (containing probe from a previous northern blot experi-
ment) is going to be used, incubate it at 68 °C for 10 min just
before adding it to the hybridization tube with the membrane.
9. Hybridization step: immediately after the 30 min prehybridiza-
tion step (step 11), replace the prehybridization solution in
the hybridization tube with the 6 ml hybridization solution
prepared in step 8. Incubate, rotating slowly, at 50 °C over-
night (16–18 h).
1. After the hybridization is complete, save the hybridization
solution at −20 °C.
2. Keep membrane in the hybridization tube and wash twice with
100 ml Low Stringency Buffer (high salt concentration and
low temperature) at room temperature for 5 min each time.
3. Preheat High Stringency Buffer (low salt concentration and
high temperature) at 50 °C.
4. Remove used Low Stringency Buffer and immediately add
preheated High Stringency Buffer.
5. Wash with 100 ml High Stringency Buffer at 50 °C twice
for 15 min each time.
Probe–target hybrids are detected with an enzyme-linked immu-
noassay, which may be more sensitive than radioactive detection
procedures when the optimal experimental conditions are followed
[4, 5]. Prepare the following solutions using DIG Wash and Block
Buffer kit just before starting the detection:
● 500 ml 1× washing buffer: 50 ml 10× Washing Buffer (shake
vigorously before use)+450 ml double-distilled RNAse-free
water.
● 50 ml 1× maleic acid buffer: 5 ml 10× Maleic Acid Buffer+45 ml
double-distilled RNAse-free water.
● 20 ml 1× blocking solution: 2 ml 10× Blocking Solution+18 ml
1× Maleic Acid buffer.
● 20 ml 1× detection buffer: 2 ml 10× Detection Buffer+18 ml
double-distilled RNAse-free water.
1. Equilibrate the membrane in a clean tray (treated with anti-
RNasespray)containing50ml1×WashingBufferfor1–5min.
2. First wash the hybridization tube with soap and water,
then treat it with RNAse Zap (Applied Biosystems,
Carlsbad, CA) and rinse with RNAse-free water.
3.4.3 Washes
3.4.4 Detection
Marc R. Reboll et al.](https://image.slidesharecdn.com/2464969-250609220113-c1bac12a/85/Rna-Mapping-Methods-And-Protocols-1st-Edition-M-Lucrecia-Alvarez-29-320.jpg)
![19
M. Lucrecia Alvarez and Mahtab Nourbakhsh (eds.), RNA Mapping: Methods and Protocols, Methods in Molecular Biology,
vol. 1182, DOI 10.1007/978-1-4939-1062-5_2, © Springer Science+Business Media New York 2014
Chapter 2
Rapid Mapping of RNA 3′ and 5′ Ends
Victoria Zismann and Mahtab Nourbakhsh
Abstract
In recent years, an enormous progress has been made in applied genomics leading to identification and
isolation of novel cDNAs. However, most attempts result in the acquisition of transcribed sequences that
represent only a part of the mRNA’s complete sequence. Rapid Amplification of cDNA Ends (RACE) is a
technique used in molecular biology to obtain the full length sequence of an RNA transcript found within
a cell. Since the first report of this technique, many significant improvements have been made on the basic
approach. This chapter describes the most recent update of the relatively simple and versatile classic RACE
protocol.
Key words Gene expression, mRNA mapping, Lambda cDNA library, cDNA end amplification,
RACE amplification
1 Introduction
Rapid Amplification of cDNA Ends (RACE) is used to extend
partial cDNAs by amplifying the 5′ or 3′ sequences of the corre-
sponding mRNAs using gene-specific primers [1, 2]. The tech-
nique requires knowledge of only a small region of the sequence
within the known partial cDNA (Fig. 1a). The first step begins
with cDNA synthesis using cellular RNA. During this step, the
Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV
RT) utilizes terminal transferase activity to add three to five resi-
dues to the 3′ end of the polymerized cDNA upon reaching the
end of an RNA template. An additional primer, which contains a
terminal stretch of annealing residues, is added to the reaction and
serves as an extended template for reverse transcription. MMLV
RT switches templates from the mRNA molecule to the primer
generating a complete cDNA copy of the original RNA with the
additional end sequence. This step is called Switching Mechanism
at 5′ End of RNA Template (SMART). The relationship of the
primers used in the SMART RACE reactions to the template and
resulting RACE products is shown in detail in Fig. 1a.](https://image.slidesharecdn.com/2464969-250609220113-c1bac12a/85/Rna-Mapping-Methods-And-Protocols-1st-Edition-M-Lucrecia-Alvarez-34-320.jpg)
![23
24. Air-dry DNA pellet for 10–15 min.
25. Add 20–50 μl of Buffer NE and resuspend the pellet by
vortexing.
26. Elute DNA by incubating the sample at room temperature for
10–15 min.
27. Centrifuge the sample at 10,000×g for 30 s at room tempera-
ture, and then transfer the supernatant containing the purified
DNA to a fresh tube. The isolated fragment(s) can now be
directly cloned into a T/A-type PCR cloning vector (e.g., Life
Technologies).
This approach relies on amplification of a cDNA sequences from
commercially available human cDNA libraries which represent the
total RNA from a cell line or tissue. The main advantage of premade
libraries is that they contain high-quality, full-length cDNAs inserted
into a self-replicating lambda vector. Once the sequence is available
in the form of a cDNA library, individual processed segments of
the original cDNA can be isolated and examined with relative ease.
3.2 Identification
of cDNA Ends Using
Premade cDNA
Libraries
Table 1
Troubleshooting guide for SMART RACE cDNA Amplification kita
Problem Recommendations
5′ or 3′-RACE
product is not
the expected
size or is absent
The cause may be a GCrich template. Use Clontech Mixes for efficient
amplification of GC-rich templates. PCR parameters may need to be
optimized for these templates
No amplified 3′ or
5′ products after
the minimum
number of
cycles at 68 °C
Return tube(s) to the thermal cycler and run five additional cycles. If the
product still does not appear, add an additional three to five cycles at
68 °C. If you are still unsuccessful, run a new PCR experiment, changing
the annealing temperature in the third set of cycles from 68 °C to
65 °C. This last program is especially useful if Tm close to 70 °C
Neither 3′ nor 5′
amplified
products
Check the quality of first-strand cDNA (if generated from poly A+RNA) using
a 32P-labeling procedure. Repeat the first-strand synthesis, substituting 1 μl
of 0.1 μCi/μl [α-32P] dATP or dCTP for 1 μl of water. Run the reaction
products on an alkaline agarose gel, and examine the banding pattern by
autoradiography. If the first-strand reaction was successful, you should see a
banding pattern similar to that produced by your RNA. Mammalian poly
A+RNA typically produces a smear from 0.5 to 12 kb. Mammalian total
RNA usually exhibits two bright bands at 1.9 kb and 4.5 kb
Multiple 5′- and/
or 3′-RACE
products
By multiple fragments you can generally start with the largest fragment from
each RACE reaction, because it is most likely to be a true, complete RACE
product. However, in the long run you should try to eliminate nonspecific
fragments by troubleshooting the reactions
a
The information in this table was partially taken and modified from SMART RACE cDNA Amplification kit manual
from Clontech; Mountain View, CA
Mapping RNA Ends](https://image.slidesharecdn.com/2464969-250609220113-c1bac12a/85/Rna-Mapping-Methods-And-Protocols-1st-Edition-M-Lucrecia-Alvarez-38-320.jpg)
![27
M. Lucrecia Alvarez and Mahtab Nourbakhsh (eds.), RNA Mapping: Methods and Protocols, Methods in Molecular Biology,
vol. 1182, DOI 10.1007/978-1-4939-1062-5_3, © Springer Science+Business Media New York 2014
Chapter 3
Single Nucleotide Mapping of RNA 5′ and 3′ Ends
Mahtab Nourbakhsh
Abstract
Nuclease protection assay is a sensitive method for detection, quantitation, and mapping of a specific RNA
in an extremely heterogeneous mixture of RNAs, such as total cellular RNA. The assay is based on a small
volume solution hybridization of a single-stranded synthetic antisense and labeled RNA probe to a RNA
sample. Thus, it is much more efficient than the common immobilized hybridization on a membrane,
such as in northern-blot analysis. After solution hybridization, different nucleases are used to remove any
remaining single-stranded nucleotides within the probe and sample RNA by digestion. Then, the remaining
probe-target hybrids are purified and separated on a denaturing polyacrylamide gel. Using a radioactive
labeled probe, the protected probe can be visualized by direct autoradiography and the copy number can
be calculated based on the specific radioactivity of the RNA probe and the length of protected fragment.
Because of its high sensitivity and resolution, nuclease protection assay is the most effective procedure for
mapping internal and external boundaries in mRNA compared to other RNA detection methods such as
RT-PCR.
Key words Intron, 5′ UTR, 3′ UTR, Double stranded RNA, Hybridization, Nuclease, S1 RNA
mapping
1 Introduction
Molecular characterization of genomic sequences involves the
qualitative and quantitative analysis of possibly transcribed
sequences. Transcribed regions can be hypothetically determined
by aligning Expressed Sequence Tags (ESTs) with genome
sequences [1]. However, available ESTs databases represent only a
fraction of transcribed regions. This makes further experimental
analysis indispensable.
The current book includes three most frequently used technical
procedures for mapping, detecting, and quantifying a particular
RNA in a total RNA sample: Northern blot analysis, nuclease pro-
tection assays, and reverse transcription-polymerase chain reaction
(RT-PCR). Although each of these techniques can be used for
qualitative and quantitative RNA detection, each procedure has
decisive advantages and/or limitations. Northern-blot analysis is](https://image.slidesharecdn.com/2464969-250609220113-c1bac12a/85/Rna-Mapping-Methods-And-Protocols-1st-Edition-M-Lucrecia-Alvarez-41-320.jpg)
![28
the only method that can provide information about the size of
the mature transcripts. RT-PCR is the most convenient, fast, and
sensitive method for comparative detection and estimation of rel-
ative abundance of transcripts in different samples. The principle
of nuclease protection assays is based on hybridization of a labeled
synthetic antisense RNA probes to a target RNA which forms a
double-stranded RNA fragment protected against specific single
strand nucleases (see Fig. 1). Following nuclease treatment and
removing of single-stranded sequences, the length and the copy
number of protected probe fragments correspond to the bound-
aries and number of target RNA in a sample. As truncation of
the full-length probe is the indicative step, it is crucial to use a
probe which overspans the possible homology region. In most
cases, probes are designed to convey unrelated vector sequences
(see Fig. 1).
The potential use of different antisense probes in nuclease pro-
tection experiments allows for simultaneous quantification of dif-
ferent RNA species in a single sample [2]. Thus, several different
target RNAs or several different fragments of a single target RNA
can be simultaneously detected in samples. First, this approach can
Fig. 1 Nuclease protection assay principle.The method is based on hybridization
of a labeled synthetic antisense RNA probes to a target RNA which forms a dou-
ble-stranded RNA fragment. S1 or similar nucleases remove single-stranded
sequences. The remaining fragment signal corresponds to the length and the
copy number of a target RNA. Digestion of the full-length probe is an indicative
step. Therefore, unspecific vector sequences should be co-transcribed which
overspans a possible homology region
Mahtab Nourbakhsh](https://image.slidesharecdn.com/2464969-250609220113-c1bac12a/85/Rna-Mapping-Methods-And-Protocols-1st-Edition-M-Lucrecia-Alvarez-42-320.jpg)
![29
discriminate between closely related targets if probes are designed
to span the regions where the related genes differ at the most.
Second, probes can be designed to protect fragments of different
sizes of target RNAs. Nuclease protection assay is thereby suitable
for precise single nucleotide mapping of external and internal junc-
tions in RNA including transcription initiation or termination sites
as well as intron and exon boundaries. This method is an important
tool for analysis of alternative splicing [3].
Although DNA can serve as a probe as well, RNA probes are
mostly preferred because RNA–RNA duplexes are more stable
than RNA–DNA duplexes in solution hybridization. The antisense
RNA probe is usually synthesized using available in vitro
Transcription kits and radioactive nucleotides. This step requires a
DNA template containing antisense sequence downstream to a
synthetic promoter, such as T7. Although end-labeling of RNA
probe is feasible as well, incorporation of numerous radioactive
nucleotides during transcription reaction reveals a probe with sig-
nificantly higher specific activity. Using the MAXIscript Kit
described in this chapter, radiolabeled RNA probes can be synthe-
sized in a 10 min reaction. The Kit can be used to incorporate any
labeled nucleotide into RNA using Sp6, T3 or T7 polymerases.
Following in solution hybridization of RNA target and anti-
sense probe a nuclease treatment removes all single-stranded
sequences, precisely at the nucleotide 3′ and 5′ to double stranded
sequence. The last stage of the assay involves the separation of pro-
tected probe and RNA fragments in a denaturing gel. A short gel
run (15 cm) is sufficient for most quantification purposes. For pre-
cise mapping experiments and exact determination of the size of
the protected fragments, they may be resolved on a denaturing
sequencing gel (60 cm) in combination with a RNA marker or
“sequencing ladder” reaction [4].
This chapter provides the complete RNase protection assay
protocol including support protocols for synthesis of labeled
probes, and quantitation and mapping of target mRNA.
2 Materials
1. MAXIscript®
kit (Life Technologies).
2. DNA template (see Note 1).
3. Labeled nucleotide (see Note 2).
4. Trichloroacetic acid: molecular biology grade.
5. Ethanol: ACS reagent grade.
6. 0.5 M EDTA.
7. Centri-Spin™ 40 Columns (Life Technologies).
2.1 Synthesis of RNA
Antisense Probe
Single Nucleotide RNA Mapping](https://image.slidesharecdn.com/2464969-250609220113-c1bac12a/85/Rna-Mapping-Methods-And-Protocols-1st-Edition-M-Lucrecia-Alvarez-43-320.jpg)
![35
M. Lucrecia Alvarez and Mahtab Nourbakhsh (eds.), RNA Mapping: Methods and Protocols, Methods in Molecular Biology,
vol. 1182, DOI 10.1007/978-1-4939-1062-5_4, © Springer Science+Business Media New York 2014
Chapter 4
Analysis of RNA Secondary Structure
Mahtab Nourbakhsh
Abstract
RNA has different levels of structural organization. The primary structure is the linear order of the
nucleotide monomers, the RNA sequence. During transcription process, the partially synthesized RNA is
folded by base-pairing and thermodynamic intramolecular or intermolecular interactions. This results in a
dynamic spreading of a secondary structure along the length of the transcribed section of the RNA. The
analysis of both primary or secondary structures requires the RNA end-labeling either at its 5′ end using a
kinase reaction with [gamma-32P]ATP, or at its 3′ end using an RNA ligation reaction with [32P]pCp.
End-labeled RNAs are then gradually breakdown using hydrolysing chemicals or a variety of enzymes
targeting specific RNA sequences and secondary structure. The most commonly used enzymes are RNase
A, T1, and V1. The partial digestion of the RNA reveals a mix of truncated RNA fragments of different
lengths, called RNA ladder. The products are then separates through a high resolution gel system and
subjected to autoradiographic analysis. Each visible fragment is labeled at one end, but comprises an
enzyme specific sequence at the other end. Final comparison of the detected RNA ladders reveals a hypo-
thetical model of the secondary RNA structure under assay conditions.
Key words Secondary structure, Double-stranded RNA, Single-stranded RNA, RNAse, Mfold
1 Introduction
RNA molecules possess a variety of single-stranded and double-
stranded regions that lead to complex three-dimensional structures.
These structures are mostly crucial for the molecule’s interactions
with other regulatory molecules like nucleic acids and proteins.
Thus, RNA structure plays a central role in many cellular processes,
including transcription initiation, elongation and termination, reg-
ulation of gene expression, and protein translation. Thus, elucidat-
ing the mechanistic aspects of RNA interactions often requires a
detailed understanding of the underlying RNA structure.
Analysis of RNA structure is traditionally based on successive
enzymatic cleavage of folded RNA using specific RNases. First,
RNA of interest needs to be synthesized in vitro using available
in vitro transcription systems. This step requires a DNA template
containing the RNA sequence of interest localized downstream to](https://image.slidesharecdn.com/2464969-250609220113-c1bac12a/85/Rna-Mapping-Methods-And-Protocols-1st-Edition-M-Lucrecia-Alvarez-49-320.jpg)
![36
a synthetic promoter, T3, T7, or SP6 (see Note 1). Using the
MAXIscript Kit described in this chapter, high amount of RNA can
be synthesized in a 10-min reaction using corresponding
polymerase. Next, the RNA product needs to be labeled at 5′ or 3′
end using a kinase reaction with [gamma-32P]ATP or an RNA
ligation reaction with [32P]pCp, respectively.
T4 Polynucleotide Kinase (PNK) catalyzes the transfer of the
gamma-phosphate of ATP to the 5′-hydroxyl termini of RNA.
This phosphate transfer is commonly referred to as a kinase or
phosphorylation reaction. RNAs with a 5′-hydroxyl (OH) group
can be added directly to a kinase reaction. However, RNAs with a
5′-phosphate should be dephosphorylated first using Calf Intestinal
Phosphatase prior to labeling with PNK and [gamma-32P]ATP.
T4 RNA ligase catalyzes the ligation of the 5′ phosphate termi-
nus of a nucleic acid donor to the 3′ OH terminus of a nucleic acid
acceptor. The reaction is ATP dependent, and the 3′ end of target
RNA is labeled by adding [32P]pCp to the reaction.
The most commonly used RNases for performing RNA
structural analysis are RNases A, V1, and T1. These enzymes bind
to specific sequences and cleave folded RNA at specific sequence
patterns. RNase A is a pancreatic ribonuclease that cleaves the
target RNA 3′ of single-stranded C and U residues. It cleaves
the phosphodiester bond between the 5′-ribose of a nucleotide
and the phosphate group attached to the 3′-ribose of an adjacent
pyrimidine nucleotide. The resulting 2′, 3′-cyclic phosphate is
hydrolysed to corresponding 3′-nucleoside phosphate. RNase V1
is a metal-dependent ribonuclease specific for dsRNA regions of 4
nucleotides or more. Cleavage occurs between the 3′-hydroxyl of
any ribonucleotide and the 5′-phosphate group of the adjacent
ribonucleotide. Commercially available RNase T1 is isolated by a
series of purification steps from recombinant E. coli strains overex-
pressing the RNase T1 gene of Aspergillus oryzae. The purified
enzyme specifically cleaves the target RNA 3′ to single-stranded
guanosine residues, producing 3′-phosphorylated ends.
To facilitate RNA structural studies, the exposure time of RNA
to RNase is strictly limited allowing for a single cleavage per RNA
strand. The end products of RNase reactions comprise of labeled
RNA ladders which can be analyzed using high resolution gel elec-
trophoresis. To help identify the cleavage site locations, another
ladder is generated by alkaline hydrolysis which cleaves RNA strand
by single nucleotides.
In addition to the described experimental analysis here, we
recommend the use of available software for predicting RNA
structure [1–3]. Secondary structure is the set of the canonical
base pairs, and secondary structure can be predicted by compara-
tive sequence analysis in silico. The most commonly used method
is free energy minimization. The accuracy of structure prediction is
then improved either by using experimental mapping data or by
Mahtab Nourbakhsh](https://image.slidesharecdn.com/2464969-250609220113-c1bac12a/85/Rna-Mapping-Methods-And-Protocols-1st-Edition-M-Lucrecia-Alvarez-50-320.jpg)
![37
predicting a structure conserved in a set of homologous sequences.
Additionally, tertiary structure, the three-dimensional arrange-
ment of atoms, can be modeled with guidance from comparative
analysis and experimental techniques. New approaches are also
available for predicting tertiary structure.
2 Materials
1. MAXIscript®
kit (Life Technologies).
2. DNA template (see Note 1).
3. Ribonucleotides.
4. Trichloroacetic acid: molecular biology grade.
5. Ethanol: ACS reagent grade.
6. 0.5 M EDTA.
7. Centri-Spin™ 40 Columns (Life Technologies).
8. T4 RNA Ligase (Ambion Cat #2140).
9. 10× T4 RNA Ligase Buffer (supplied with Ambion’s T4 RNA
Ligase: 0.5 M Tris–HCl, pH 7.8, 0.1 M MgCl2, 0.1 M DTT,
10 mM ATP).
10. [32P]pCp.
11. RNase-free Sephadex G-25 or G-50 spin columns such as
Ambion’s NucAway™ Spin Columns (Cat #10070).
1. Nuclease-free Water.
2. 10× Dephosphorylation buffer (0.5 M Tris–HCl, pH 8.5,
1 mM EDTA, pH 8).
3. Calf Intestinal phosphatase (CIP; 0.1 U/μl).
4. Phosphatase removal reagent (available in the KinaseMax™ Kit).
5. [gamma-32P]ATP (7,000 Ci/mmol, 150 mCi/ml).
6. 10× Kinase buffer (500 mM Tris, pH 7.5, 100 mM MgCl2,
50 mM DTT).
7. T4 Polynucleotide Kinase (10 U/ml).
8. RNase-free Sephadex G-25 or G-50 spin columns such as
Ambion’s NucAway™ Spin Columns (Cat #10070).
1. 0.1–3 μg end-labeled RNA.
2. Yeast RNA (10 mg/ml, Ambion Cat # 7118).
3. 1× Alkaline Hydrolysis Buffer (supplied with Ambion’s RNA
Grade Ribonucleases: 50 mM Sodium Carbonate [NaHCO3/
Na2CO3] pH 9.2, 1 mM EDTA).
2.1 RNA Synthesis
2.2 RNA 3′ End
Labeling
2.3 RNA 5′ Labeling
2.4 RNA Hydrolysis
RNA Structure Analysis](https://image.slidesharecdn.com/2464969-250609220113-c1bac12a/85/Rna-Mapping-Methods-And-Protocols-1st-Edition-M-Lucrecia-Alvarez-51-320.jpg)
![39
1. Combine 2 μl 10× T4 RNA Ligase Buffer, 50–100 pmol
RNA, equimolar amount (50–100 pmol) [32P]pCp and
RNase-free water to a final volume of 18 μl in a single RNase-
free microfuge tube.
2. Mix thoroughly by pipetting the mixture up and down gently.
3. Add 2 μl T4 RNA Ligase (10 U) and mix thoroughly by pipet-
ting the mixture up and down gently.
4. Incubate at 4 °C overnight (10–12 h).
5. Remove unincorporated nucleotides by applying the mixture
to an RNase-free Sephadex G-25 or G-50 spin column (e.g.,
NucAway Spin Columns) following the manufacturer’s
recommendations.
6. If the RNA is not labeled efficiently, follow the instructions in
Table 1.
1. Combine Nuclease-free Water to make a final volume of
10 μl, 0.1–10 pmol RNA, 1 μl 10× dephosphorylation buffer
(0.5 M Tris–HCl, pH 8.5, 1 mM EDTA, pH 8) and 1 μl Calf
Intestinal Phosphatase (CIP; 0.1 U/μl) in a single RNase-
free microfuge tube.
2. Mix thoroughly by pipetting the mixture up and down gently.
3. Incubate for 1 h at 37 °C.
4. Remove the Calf Intestine Alkaline Phosphatase by use of the
Phosphatase Removal Reagent (available in the KinaseMax™ Kit).
5. Combine nuclease-free water to make a final volume of 20 μl,
25 pmol [gamma-32P]ATP (7,000 Ci/mmol, 150 mCi/ml),
2 μl 10× Kinase Buffer (500 mM Tris, pH 7.5, 100 mM MgCl2,
50 mM DTT), and 1 μl T4 Polynucleotide Kinase (10 U/ml).
6. Mix thoroughly by pipetting the mixture up and down gently.
7. Incubate at 37 °C for 1 h.
8. Remove unincorporated nucleotides by applying the mixture
to an RNase-free Sephadex G-25 or G-50 spin column (e.g.,
NucAway Spin Columns) following the manufacturer’s
recommendations.
9. If the RNA is not labeled efficiently, follow the instructions in
Table 1.
This procedure provides a gel electrophoresis “ladder” of hydro-
lyzed RNA fragments. In the procedure, three different hydrolysis
times are used. After the experiment, select the ladder that pro-
vides the best distribution of nucleic acids over the range of lengths
needed for your experiments.
1. Mix 0.1–3 μg of end-labeled RNA and 3 μg of yeast tRNA in
5 μl or less.
3.2 RNA 3′ End
Labeling
3.3 RNA 5′ End
Dephosphorylation
and 5′ End Labeling
3.4 RNA Secondary
Structure Analysis
3.4.1 Alkaline Hydrolysis
RNA Structure Analysis](https://image.slidesharecdn.com/2464969-250609220113-c1bac12a/85/Rna-Mapping-Methods-And-Protocols-1st-Edition-M-Lucrecia-Alvarez-53-320.jpg)
![The right to construe the constitution he argued from the principles
advanced by Mr. Madison, in the debate on the power of removability,
and read sundry observations from Lloyd's Register, made by that
gentleman, corroborative of this sentiment. Those arguments, he
conceived, applied very aptly to the present subject.
Matters of a fiscal nature necessarily devolve on the General
Government, and he urged that every power resulting from the
acknowledged right of Congress to control the finances of this
country must be as necessarily implied as in the case of the power
of removability.
He then alluded to the expediency of a National Bank. The Secretary
gave notice, in his first report, that this plan was in contemplation.
Nothing was ever read with greater avidity; and though it is now
more than a year since this intimation was given, yet no objections
have been offered against it either by the States or by individuals—
even the State of North Carolina has not mentioned it. [Here Mr.
Bloodworth (if the reporter did not misunderstand) informed Mr.
Smith that the report had not been seen by the Legislature of North
Carolina.] Mr. Smith said he was sorry for it—and then proceeded to
notice some partial quotations, made by Mr. Jackson, from Dr. Smith's
Wealth of Nations, against bank systems. He said, he could have
wished the gentleman had been more copious in his quotations from
that author; if he had, he would have found that that author has
fully demonstrated their utility.
He noticed the divisions of opinions on the subject of a National
Bank in the city of Philadelphia. He supposed ideas of personal
advantages induced these opposing sentiments. He, however,
thought this subject should be taken up altogether on general
principles; and even if its immediate influence should not extend to
the extremes of the Union, if the establishment promises a general
preponderating advantage, local considerations must be considered
in a secondary point of view. The principal inquiry is, will the
institution facilitate the management of the finances? This, he
thought, had been made apparent. This is the opinion of the](https://image.slidesharecdn.com/2464969-250609220113-c1bac12a/85/Rna-Mapping-Methods-And-Protocols-1st-Edition-M-Lucrecia-Alvarez-76-320.jpg)
![individuals and of States, had been explained by a gentleman from
Virginia, (Mr. Madison.) He should only remark, that all those who
opposed the Government dreaded this doctrine; those who
advocated it, declared that it could not be resorted to; and all
combined in opinion that it ought not to be tolerated. Never did any
country more completely unite in any sentiment than America in this,
"that Congress ought not to exercise, by implication, powers not
granted by the constitution." And is it not strange? For the admission
of this doctrine destroys the principle of our Government at a blow;
it at once breaks down every barrier which the Federal constitution
had raised against unlimited legislation. He said, that necessity was
the most plausible pretext for breaking the spirit of the social
compact, but the people of this country have anticipated that
pretext. They have said to the Ministers of this country, "we have
given you what we think competent powers, but if experience proves
them inadequate, we will enlarge them; but, in the mean time, dare
not usurp those which we have reserved."
It is agreed on all hands, that the power to incorporate the
subscribers to a banking company, is not expressly granted, and
although gentlemen have agreed that it is implied—that it is an
incident, that it is a means for effectuating powers expressly
granted, yet they are not agreed as to the particular power to which
this is an incident. They admit, that the sweeping clause in the
constitution confers no additional power. But if he understood the
gentlemen, several of them were of opinion that all governments,
instituted for certain ends, draw to them the means of execution as
of common right. This doctrine would make ours but a short
constitution. [Here he read the preamble and then said:] Here is
your constitution! Here is your bill of rights! Do these gentlemen
require any thing more respecting the powers of Congress, than a
description of the ends of government? And if, of right, they can
carry these into effect, will they regard the means, though they be
expressly pointed out? But I would ask if there is any power under
heaven which could not be exercised within the extensive limits of
this preamble?](https://image.slidesharecdn.com/2464969-250609220113-c1bac12a/85/Rna-Mapping-Methods-And-Protocols-1st-Edition-M-Lucrecia-Alvarez-79-320.jpg)
Rna Mapping Methods And Protocols 1st Edition M Lucrecia Alvarez
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- 5. RNA Mapping M. Lucrecia Alvarez Mahtab Nourbakhsh Editors Methods and Protocols Methods in Molecular Biology 1182
- 6. ME T H O D S I N MO L E C U L A R BI O LO G Y Series Editor John M. Walker School of Life Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK For further volumes: http://www.springer.com/series/7651
- 8. RNA Mapping Methods and Protocols Edited by M. Lucrecia Alvarez Diabetes, Cardiovascular, and Metabolic Diseases, Translational Genomics Research Institute,Phoenix, AZ, USA Mahtab Nourbakhsh DepartmentofPharmacyandBiotechnology,GermanUniversityinCairo,Berlin,Germany DepartmentofPlasticSurgery,UniversityHospitaloftheRWTHAachen,Aachen,Germany
- 9. ISSN 1064-3745 ISSN 1940-6029 (electronic) ISBN 978-1-4939-1061-8 ISBN 978-1-4939-1062-5 (eBook) DOI 10.1007 /978-1-4939-1062-5 Springer New York Heidelberg Dordrecht London Library of Congress Control Number: 2014942861 © Springer Science+Business Media New York 2014 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. Exempted from this legal reservation are brief excerpts in connection with reviews or scholarly analysis or material supplied specifically for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work. Duplication of this publication or parts thereof is permitted only under the provisions of the Copyright Law of the Publisher’s location, in its current version, and permission for use must always be obtained from Springer. Permissions for use may be obtained through RightsLink at the Copyright Clearance Center. Violations are liable to prosecution under the respective Copyright Law. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. While the advice and information in this book are believed to be true and accurate at the date of publication, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein. Printed on acid-free paper Humana Press is a brand of Springer Springer is part of Springer Science+Business Media (www.springer.com) Editors M. Lucrecia Alvarez Diabetes, Cardiovascular, and Metabolic Diseases Translational Genomics Research Institute Phoenix, AZ, USA Mahtab Nourbakhsh Department of Pharmacy and Biotechnology German University in Cairo Berlin, Germany Department of Plastic Surgery University Hospital of the RWTH Aachen Aachen, Germany
- 10. v About 40 years ago, scientists believed that only less than 3 % of human DNA encodes proteins. The remaining 97 % was labeled as “genetic junk”—DNA that is never transcribed and has no biological functions—and attributed to molecular trial-and-error that has accu- mulated over the course of evolution. However, it has recently become apparent that more than 85 % of the genome—including most of the non-protein-coding DNA—is indeed transcribed and performs essential biological functions. In fact, the Encyclopedia of DNA Elements (ENCODE) Project has recently claimed that at least 80 % of the human genome has a biochemical function. Therefore, the existence of “junk DNA” is gradually becoming a myth. Nowadays, the continuous discovery of a myriad of non-protein-coding RNAs with regulatory functions, such as microRNAs and long noncoding RNAs, contributes to the unraveling of the remarkable versatility of the RNA molecule. However, the discovery of new functional transcripts is yet to come. The recent developments in RNA-based technologies in concert with the increasing importance of RNA molecules as biomarkers in diagnostics and therapeutics call for a sum- mary of both modern and traditional strategies for characterization of cellular RNAs. “RNA Mapping: Methods and Protocols” intends to provide not only an update of many of the classic techniques but also an introduction, description, and summary of newer approaches that go beyond the pure biomedical applications. This book is particularly targeted to biochemists, molecular biologists, and any researcher in the life sciences interested in the molecular characterization of coding and noncoding RNAs. The purpose of “RNA Mapping: Methods and Protocols” is therefore to provide instruction and inspiration for all those scientists who are facing the challenges of the discovery and/or functional character- ization of RNA molecules for a wide variety of applications ranging from novel biomedical diagnostics to therapeutics and biomaterials. The book has been organized in two separate parts: Part I contains 13 protocols for the structural mapping of cellular RNAs. These chap- ters encompass the definition of RNA boundaries, primary, secondary, and tertiary RNA structure as well as RNA identification and quantification protocols based on structural properties of target RNAs. For example, step-by-step descriptions of different methods for RNA profiling using quantitative real-time PCR or next-generation sequencing have been included in this section. In Part II, 15 protocols focus on a variety of functional elements in RNA including the mapping of internal ribosome entry sites and regulatory mRNA elements as well as the identification of actively translated mRNAs. In addition, this section describes methods for the identification of novel RNA functions with a special focus on microRNA targets predic- tion and their experimental validation using state-of-the-art technology. The collection of protocols in this volume is a consequence of the emerging interest in the characterization of cellular RNAs urged by their potential use as diagnostic biomarkers or therapeutic targets. In particular, the biological relevance of microRNAs in human Preface
- 11. vi physiology and disease development is highlighted in the 16 chapters focused on methods for their physical and functional mapping. We would like to thank all the contributing authors for providing excellent chapters and sharing years of their experience in a specific technique, to John Walker for editorial guidance, and to the staff of Humana Press for professional production of this volume. Phoenix, AZ, USA M. Lucrecia Alvarez Aachen, Germany Mahtab Nourbakhsh Preface
- 12. vii Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix PART I STRUCTURAL RNA MAPPING 1 Full-Length Characterization of Transcribed Genomic Regions . . . . . . . . . . . . 3 Marc R. Reboll, M. Lucrecia Alvarez, and Mahtab Nourbakhsh 2 Rapid Mapping of RNA 3′ and 5′ Ends. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Victoria Zismann and Mahtab Nourbakhsh 3 Single Nucleotide Mapping of RNA 5′ and 3′ Ends. . . . . . . . . . . . . . . . . . . . . 27 Mahtab Nourbakhsh 4 Analysis of RNA Secondary Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 Mahtab Nourbakhsh 5 Tertiary Structure Mapping of the Pri-miRNA miR-17~92 . . . . . . . . . . . . . . . 43 Steven G. Chaulk and Richard P. Fahlman 6 In Situ Hybridization Detection of miRNA Using LNA™ Oligonucleotides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 Stefania Cotta Doné and Olga Beltcheva 7 Quantification of miRNAs by a Simple and Specific qPCR Method . . . . . . . . . 73 Susanna Cirera and Peter K. Busk 8 RNA Isolation for Small RNA Next-Generation Sequencing from Acellular Biofluids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 Kasandra L. Burgos and Kendall Van Keuren-Jensen 9 Sequencing Small RNA: Introduction and Data Analysis Fundamentals . . . . . . 93 Jai Prakash Mehta 10 Measuring Expression Levels of Small Regulatory RNA Molecules from Body Fluids and Formalin-Fixed, Paraffin-Embedded Samples . . . . . . . . 105 Adrienn Gyongyosi, Otto Docs, Zsolt Czimmerer, Laszlo Orosz, Attila Horvath, Olga Török, Gabor Mehes, Laszlo Nagy, and Balint L. Balint 11 MicroRNA Profiling in Plasma or Serum Using Quantitative RT-PCR . . . . . . 121 Marina C. Costa, Ana Lúcia Leitão, and Francisco J. Enguita 12 MicroRNA Profiling of Exosomes Isolated from Biofluids and Conditioned Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131 Sweta Rani 13 Isolation of Urinary Exosomes for RNA Biomarker Discovery Using a Simple, Fast, and Highly Scalable Method . . . . . . . . . . . . . . . . . . . . . 145 M. Lucrecia Alvarez Contents
- 13. viii PART II FUNCTIONAL RNA MAPPING 14 Identification of Actively Translated mRNAs . . . . . . . . . . . . . . . . . . . . . . . . . . 173 Marc R. Reboll and Mahtab Nourbakhsh 15 Mapping of Internal Ribosome Entry Sites (IRES) . . . . . . . . . . . . . . . . . . . . . 179 Sarah Mehrtens and Marc R. Reboll 16 Mapping of Protein Binding RNA Elements . . . . . . . . . . . . . . . . . . . . . . . . . . 187 Marc R. Reboll 17 Purification of RNA-Binding Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195 Birgit Ritter and Marc R. Reboll 18 De Novo Approach to Classify Protein-Coding and Noncoding Transcripts Based on Sequence Composition. . . . . . . . . . . . . . . . . . . . . . . . . . 203 Haitao Luo, Dechao Bu, Liang Sun, Runsheng Chen, and Yi Zhao 19 Computational Methods to Predict Long Noncoding RNA Functions Based on Co-expression Network . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209 Yi Zhao, Haitao Luo, Xiaowei Chen, Yi Xiao, and Runsheng Chen 20 MicroRNA Biogenesis: Dicing Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219 Carlos A. Melo and Sonia A. Melo 21 Faster Experimental Validation of microRNA Targets Using Cold Fusion Cloning and a Dual Firefly-Renilla Luciferase Reporter Assay . . . . . . . 227 M. Lucrecia Alvarez 22 Experimental Validation of Predicted Mammalian MicroRNAs of Mirtron Origin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245 Anita Schamberger and Tamás I. Orbán 23 A Guide for miRNA Target Prediction and Analysis Using Web-Based Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265 Ana Lúcia Leitão, Marina C. Costa, and Francisco J. Enguita 24 Tapping MicroRNA Regulation Networks Through Integrated Analysis of MicroRNA–mRNA High-Throughput Profiles . . . . . . . . . . . . . . . 279 Anthony D. Saleh and Hui Cheng 25 miRWalk Database for miRNA–Target Interactions . . . . . . . . . . . . . . . . . . . . . 289 Harsh Dweep, Norbert Gretz, and Carsten Sticht 26 A Schematic Workflow for Collecting Information About the Interaction Between Copy Number Variants and MicroRNAs Using Existing Resources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307 Harsh Dweep, Norbert Gretz, and Kyriakos Felekkis 27 SYBR® Green and TaqMan® Quantitative PCR Arrays: Expression Profile of Genes Relevant to a Pathway or a Disease State . . . . . . . . . . . . . . . . 321 M. Lucrecia Alvarez and Stefania Cotta Doné 28 Comprehensive Meta-analysis of MicroRNA Expression Using a Robust Rank Aggregation Approach. . . . . . . . . . . . . . . . . . . . . . . . . . 361 Urmo Võsa, Raivo Kolde, Jaak Vilo, Andres Metspalu, and Tarmo Annilo Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 375 Contents
- 14. ix M. LUCRECIA ALVAREZ • Diabetes, Cardiovascular, and Metabolic Diseases, Translational Genomics Research Institute, Phoenix, AZ, USA TARMO ANNILO • Estonian Genome Center, University of Tartu, Tartu, Estonia BALINT L. BALINT • Department of Biochemistry and Molecular Biology, Center for Clinical Genomics and Personalized Medicine, University of Debrecen Medical and Health Science Center (UD MHSC), Debrecen, Hungary OLGA BELTCHEVA • Department of Medical Chemistry and Biochemistry, Molecular Medicine Center, Medical University of Sofia, Sofia, Bulgaria DECHAO BU • Key Laboratory of Intelligent Information Processing, Institute of Computing Technology, Chinese Academy of Sciences, Beijing, Republic of China KASANDRA L. BURGOS • Neurogenomics Division, Translational Genomics Research Institute, Phoenix, AZ, USA PETER K. BUSK • Department of Biotechnology, Chemistry and Environmental Engineering, Aalborg University Copenhagen, Copenhagen, Denmark STEVEN G. CHAULK • Department of Biochemistry, University of Alberta, Edmonton, AB, Canada RUNSHENG CHEN • Laboratory of Bioinformatics and Non-coding RNA, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China XIAOWEI CHEN • Laboratory of Bioinformatics and Non-coding RNA, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China HUI CHENG • Tumor Biology Section, Head and Neck Surgery Branch, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, MD, USA SUSANNA CIRERA • Faculty of Health and Medical Sciences, Department of Veterinary Clinical and Animal Sciences, University of Copenhagen, Frederiksberg, Denmark MARINA C. COSTA • Faculdade de Medicina, Instituto de Medicina Molecular, Universidade de Lisboa, Lisbon, Portugal ZSOLT CZIMMERER • Nuclear Receptor Research Group and MTA-DE Lendulet Immunogenomics Research Group, Department of Biochemistry and Molecular Biology, University of Debrecen Medical and Health Science Center, Debrecen, Hungary OTTO DOCS • Department of Pathology, University of Debrecen Medical and Health Science Center, Debrecen, Hungary STEFANIA COTTA DONÉ • Diabetes, Cardiovascular, and Metabolic Diseases, Translational Genomics Research Institute, Phoenix, AZ, USA HARSH DWEEP • Medical Faculty Mannheim, Medical Research Center, University of Heidelberg, Mannheim, Germany FRANCISCO J. ENGUITA • Faculdade de Medicina, Instituto de Medicina Molecular, Universidade de Lisboa, Lisbon, Portugal RICHARD P. FAHLMAN • Department of Biochemistry, University of Alberta, Edmonton, AB, Canada KYRIAKOS FELEKKIS • Department of Biological Sciences, University of Nicosia, Nicosia, Cyprus Contributors
- 15. x NORBERT GRETZ • Medical Faculty Mannheim, Medical Research Center, University of Heidelberg, Mannheim, Germany ADRIENN GYONGYOSI • Department of Biochemistry and Molecular Biology, Center for Clinical Genomics and Personalized Medicine, University of Debrecen Medical and Health Science Center, Debrecen, Hungary ATTILA HORVATH • Department of Biochemistry and Molecular Biology, Center for Clinical Genomics and Personalized Medicine, University of Debrecen Medical and Health Science Center, Debrecen, Hungary KENDALL VAN KEUREN-JENSEN • Neurogenomics Division, Translational Genomics Research Institute, Phoenix, AZ, USA RAIVO KOLDE • Institute of Computer Science, University of Tartu, Tartu, Estonia ANA LÚCIA LEITÃO • Faculdade de Ciências e Tecnologia, Departamento de Ciências e Tecnologia da Biomassa, Universidade Nova de Lisboa, Caparica, Portugal HAITAO LUO • Key Laboratory of Intelligent Information Processing, Institute of Computing Technology, Chinese Academy of Sciences, Beijing, Republic of China GABOR MEHES • Department of Pathology, University of Debrecen Medical and Health Science Center, Debrecen, Hungary SARAH MEHRTENS • Robert Koch Hospital, Gehrden, Germany JAI PRAKASH MEHTA • Conway Institute of Biomolecular & Biomedical Research, University College Dublin, Dublin, Ireland CARLOS A. MELO • Division of Gene Regulation, The Netherlands Cancer Institute, Amsterdam, The Netherlands; Doctoral Program in Biomedicine and Experimental Biology, Centre for Neuroscience and Cell Biology, Coimbra, Portugal SONIA A. MELO • Department of Cancer Biology, Metastasis Research Center, University of Texas MD Anderson Cancer Center, Houston, TX, USA; Genetic Dynamics of Cancer Cells, Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), Portugal ANDRES METSPALU • Department of Biotechnology, Institute of Molecular and Cell Biology, and Estonian Genome Center, University of Tartu, Tartu, Estonia LASZLO NAGY • Department of Biochemistry and Molecular Biology, Center for Clinical Genomics and Personalized Medicine, University of Debrecen Medical and Health Science Center, Debrecen, Hungary MAHTAB NOURBAKHSH • Department of Pharmacy and Biotechnology, German University in Cairo, Berlin, Germany; Department of Plastic Surgery, University Hospital of the RWTH Aachen, Aachen, Germany TAMÁS I. ORBÁN • Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary; Chemical Technology Transfer Ltd., Budapest, Hungary LASZLO OROSZ • Department of Obstetrics and Gynecology, University of Debrecen Medical and Health Science Center, Debrecen, Hungary SWETA RANI • REMEDI, National Centre for Biomedical Engineering Science (NCBES), NUI Galway, Galway, Ireland MARC R. REBOLL • Molecular and Translational Cardiology, Medical School Hannover, Hannover, Germany BIRGIT RITTER • Institute for Virology, Medical School Hannover, Hannover, Germany ANTHONY D. SALEH • Tumor Biology Section, Head and Neck Surgery Branch, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, MD, USA Contributors
- 16. xi ANITA SCHAMBERGER • Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary CARSTEN STICHT • Medical Faculty Mannheim, Medical Research Center, University of Heidelberg, Mannheim, Germany LIANG SUN • Key Laboratory of Intelligent Information Processing, Institute of Computing Technology, Chinese Academy of Sciences, Beijing, Republic of China OLGA TÖRÖK • Department of Obstetrics and Gynecology, University of Debrecen Medical and Health Science Center, Debrecen, Hungary JAAK VILO • Institute of Computer Science, University of Tartu, Tartu, Estonia URMO VÕSA • Department of Biotechnology, Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia YI XIAO • Department of Physics, Huazhong University of Science and Technology, Wuhan, China YI ZHAO • Key Laboratory of Intelligent Information Processing, Institute of Computing Technology, Chinese Academy of Sciences, Beijing, Republic of China VICTORIA ZISMANN • Translational Genomics Research Institute, Phoenix, AZ, USA Contributors
- 17. Part I Structural RNA Mapping
- 18. 3 M. Lucrecia Alvarez and Mahtab Nourbakhsh (eds.), RNA Mapping: Methods and Protocols, Methods in Molecular Biology, vol. 1182, DOI 10.1007/978-1-4939-1062-5_1, © Springer Science+Business Media New York 2014 Chapter 1 Full-Length Characterization of Transcribed Genomic Regions Marc R. Reboll, M. Lucrecia Alvarez, and Mahtab Nourbakhsh Abstract In the last years, an enormous progress has been made in the identification of genomic sequences. Given that genomic sequences can have various functions (e.g., structural organization, gene regulation, transcriptional start, and protein coding), molecular characterization is essential for progressing from the initial identification of genomic sequences to the delineation of a specific biological mechanism. Mapping of transcribed sequences is the initial step in functional characterization of genomic sequences. Northern blot analysis allows for a direct and detailed characterization of transcribed sequences, like size and splicing variants, and provides a relative comparison of transcript abundance between different cellular conditions. This method includes separation of total cellular RNA by size via gel electrophoresis, RNA transfer to a membrane, and RNA hybridization with a complementary labeled genomic probe. Key words Genomic sequences, RNA mapping, Northern blot analysis, Nonradioactive northern, Digoxigenin, DIG-labeling 1 Introduction RNA synthesis is usually catalyzed by RNA polymerase using genomic DNA as a template. Transcription initiation starts with the binding of RNA polymerase to the regulatory DNA usually found “upstream” of the protein coding region. RNA polymerase progresses along the DNA template from 3′ to 5′ end synthesizing a complementary RNA molecule. When the RNA polymerase reaches a termination sequence, it dissociates from the complex and the generation of a primary RNA is accomplished. Depending on their functions, RNAs are often modified by enzymes after tran- scription. For example, a poly(A) tail and a 5′ cap structure are added to protein coding messenger RNA (mRNA) and introns are removed by the spliceosome. However, many RNAs do not encode a protein. Indeed, about 97 % of the transcribed sequences in mammalian cells are classified as non-protein-coding sequences. Some noncoding RNAs are transcribed by their own genes, the
- 19. 4 so-called RNA genes. But some are derived from intron sequences of spliced RNAs. The most prominent noncoding RNAs are trans- fer (tRNA) or ribosomal RNAs (rRNA). They are involved in translation of mRNAs to proteins. Other non-protein-coding RNAs are involved in regulation of gene activity or RNA process- ing sequence directed digestion and ligation. To date, bioinformatics approaches have been heavily utilized to obtain a hypothetical map of a genomic sequence, but func- tional characterization of all genes will require detailed and precise mapping of unknown cellular RNAs. While there are several labo- rious methods for identification and characterization of transcribed genomic regions, this chapter will focus on the best established and most comprehensive method: Northern blot analysis using cellular mRNA. Northern blot analysis allows for a direct and detailed characterization of transcribed sequences, and can be combined with different methods to detect cellular localization, splicing variants, and relative abundance. For instance, RNA sta- bility can be monitored by blocking the overall RNA synthesis in cells treated with Actinomycin D. Thus, northern blot analysis can be used to monitor the stability of target RNA in the course of time. The first northern blot step, separation of cellular RNAs by size via gel electrophoresis, is very critical and should be adjusted to the expected RNA size. Otherwise, multiple gels need to be prepared to cover a broad range of RNAs from less than 20 nucle- otides to over 10 kilo bases. The second step, RNA transfer to a membrane, needs to be adjusted to the gel characteristics. The third step, hybridization with a complementary genomic DNA, can be performed using radioactive or nonradioactive digoxigenin (DIG) labeled DNA probe. 2 Materials 1. 10× MOPS electrophoresis buffer: 41.8 g MOPS in 700 ml of sterile DEPC-treated water. Adjust pH to 7.0 with 2 N NaOH. Add 20 ml of DEPC-treated 1 M sodium acetate and 20 ml of DEPC-treated 0.5 M EDTA (pH 8.0). Adjust vol- ume to 1 L with DEPC-treated water. 2. Sample buffer: 1× MOPS, 6.85 % formaldehyde, 50 % deion- ized formamide, 4 % ficoll 400, 0.01 % bromophenol blue, and 0.01 % xylene cyanol. 3. SYBR green II RNA gel stain or ethidium bromide. 4. Horizontal electrophoresis unit (i.e., Sigma-Aldrich Corp). 5. Whatman 3MM paper. 1. Herring sperm DNA. 2. Yeast total RNA. 2.1 Northern Blot Analysis 2.1.1 Radioactive Method Marc R. Reboll et al.
- 20. 5 3. Dextran sulfate. 4. Sodium chloride (Sigma-Aldrich Corp). 5. Hybond-N+membrane. 6. Rediprime II DNA labeling kit (GE Healthcare). 7. Illustra MicroSpin G-25 Columns (GE Healthcare). 8. Hybridization oven (i.e., Sigma-Aldrich Corp). 9. 20× SSC transfer buffer: 3 M sodium chloride, 300 mM tri- sodium citrate dehydrate, pH 7.0. 10. Buffer A: 2× SSC. 11. Buffer B: 1× SSC, 0.1 % SDS. 12. Buffer C: 0.2× SSC, 0.1 % SDS, preheat to 62 °C. 13. Buffer D: 0.1× SSC. 1. To test DIG-labeled DNA probe by agarose gel: agarose, bro- mophenol blue, xylene cyanol, SYBR® Safe DNA Gel Stain 10,000× concentrate (Invitrogen, Inc; Carlsbad, CA) or ethid- ium bromide. 2. 50× TAE (Tris-acetate-EDTA) buffer: 242 g/L Tris base, 5.7 % v/v, 0.05 M EDTA, pH 8.0. 3. Zeta probe GT membrane (Bio-Rad) or any positively charged nylon membrane. 4. UV crosslinker (Stratagene). 5. PCR DIG Probe Synthesis kit and Anti-DIG-AP antibody for detection (Roche Applied Science). 6. Oligonucleotide primers. 7. GeneAmp PCR system 9700 (Life Technologies) or any other thermocycler. 8. RNAse Zap (Life Technologies). 9. DIG East Hyb solution (Roche Applied Science). 10. Low Stringency Buffer: 2× SSC, 0.1 % (w/v) SDS. 11. High Stringency Buffer: 0.1× SSC, 0.1 % (w/v) SDS. 12. DIG block and wash buffer (Roche Applied Science). 13. CDP-star (Roche Applied Science). 14. KODAK BioMax XAR film. 3 Methods Northern blotting is a straightforward procedure that offers opportunities to evaluate progress at various points during the protocol (e.g., assessing integrity of RNA samples, evaluating efficiency of membrane transfer, and so forth). In this protocol, 2.1.2 Nonradioactive Method RNA Full-Length Characterization
- 21. 6 RNA that has been isolated from the tissue of choice is first separated by size via denaturing agarose gel electrophoresis. Following electrophoresis, RNA is transferred from the gel to a membrane by capillary action, after which it is cross-linked to the membrane for immobilization, and hybridized with a labeled probe for transcript detection. Northern blot analysis can be performed using either radiolabeled or non-isotopically labeled DNA as hybridization probes. The choice of label is generally dependent on a number of considerations, including sensitivity and resolu- tion. Radioactive DNA probes can be quickly prepared, offer rapid detection, and, importantly, provide much higher sensitivity com- pared to nonradioactive probes. For example, nucleotides labeled with 32 P enable high specific activity probes up to 2×109 dpm/μg, which can detect as little as 10 fg of target RNA, and it is widely appreciated that sensitivity can be of paramount importance when investigating novel transcripts and potential splicing variants whose cellular abundance is not yet known. However, a drawback of the radioactive method is the need for licensing as well as the safety issues, both of which may limit the feasibility of radioisotope use in the laboratory. Further, half-lives of some radioisotopes (i.e., 32 P) are quite short, so reagents must be used quickly or purchased on a regular basis. In contrast, the nonradioactive system utilizing digoxigenin (DIG) is an alternative method for detection of cellu- lar RNAs [1, 2] and offers unique advantages such as enhanced probe stability and shorter film exposure times for detection. In this chapter we describe methods for northern blot analysis that utilize both radioactive and nonradioactive probe preparation. However, the initial steps of northern blot analysis, including gel electrophoresis transfer, and immobilization of RNA to membrane are independent of the type of probe used [3]. 1. Prepare gel casting tray by sealing ends of gel chamber with tape or proper casting system. Place appropriate number of combs in gel tray. 2. Prepare 1 % (w/v) agarose in a glass flask, add water, and heat solution in a microwave until agarose is completely dissolved. For 120 ml gel volume, dissolve 1.2 g agarose in 86.4 ml water. 3. Cool agarose solution to 65–70 °C in a water bath. Swirl occa- sionally to prevent uneven cooling. 4. After cooling, add 21.6 ml formaldehyde and 12 ml of 10× MOPS to a final volume of 120 ml. 5. Add 12 μl of SYBR green II RNA gel stain to the gel mix. Alternatively, ethidium bromide can be added to the cooled agarose solution (final concentration 0.5 μg/ml) or directly to the RNA samples (see step 13). 6. Ensure that there is enough space between the bottom of the comb and the gel tray (0.5–1.0 mm) to allow for proper well formation and to avoid sample leakage (see Note 1). 3.1 Denaturing Agarose Gel Electrophoresis Marc R. Reboll et al.
- 22. 7 7. Gently mix and pour the cooled agarose solution onto the gel tray in a fume hood to a thickness of 3–5 mm, being careful to avoid generation of bubbles on the gel surface. Insert comb either before or immediately after pouring; if inserted after pouring, make sure that no bubbles form along the teeth of the comb. Let the gel set for at least 60 min. 8. Leaving the comb in the gel, place the gel in the electrophoresis tank. Fill the tank with enough 1× MOPS gel running buffer to cover the gel with approximately 1 mm of liquid above the sur- face of the gel. If too much buffer is used, the electric current will flow through the buffer instead of the gel. Carefully remove the comb from the gel, being careful not to tear wells. 9. The amount of cellular RNA required to detect a specific tran- script can vary over a wide range and strongly depends on the number of transcribed RNA molecules per cell. For radioactive northern blot analysis use 5–25 μg of total cellular RNA or 1–2 μg poly(A+) RNA in each lane. For the nonradioactive procedure, use 5 μg of total RNA. Dry down the appropriate amount of RNA in 1.5 ml tubes, if necessary, then reconstitute in buffer prior to gel loading. 10. Add DEPC-treated water to set all samples to an equal volume between 5 and 10 μl. 11. Add equal volume of sample buffer. Use a master mix of sample buffer to avoid concentration variations between the samples. 12. Denature samples 5 min at 90 °C, spin tubes for 10 s, then chill on ice. 13. If appropriate, add 0.5 μl ethidium bromide (0.5 μg/μl) to each sample (see step 5). 14. Slowly and carefully pipette samples into wells. 15. Forasmallelectrophoresischamber,performapre-electrophoresis run of 5–10 min at 55 V and a main run at 50–60 V. For a large electrophoresis chamber, do a pre-electrophoresis run for 5–10 min at 100 V and a main run at 100–120 V. Main run time depends on the size of the transcript that is going to be detected. The gel run can be adjusted by visualization of ribosomal RNA bands at any time during the run. 1. Cut a nylon membrane to the exact size of the gel. Use Hybond N+or Zeta Probe membranes for radioactive and nonradioac- tive procedures, respectively. Both Hybond N+and Zeta Probe are nylon membranes ideally suited for nucleic acids, but each has unique properties to enhance sensitivity to specific labeling techniques. 2. Cut four pieces of Whatman 3MM paper. Three of them should be the same size of the gel, and the fourth one should be twice as wide and long enough to fit under the gel and reach to the bottom of the dish on either side. 3.2 Transfer to Membrane and Immobilization RNA Full-Length Characterization
- 23. 8 3. When electrophoresis is complete, wash the agarose gel twice in water and take a photograph under ultraviolet light. The integrity and size distribution of total RNA can assessed at this point (see Note 2). 4. Prepare 10× SSC and filter for long term storage. Carefully put the gel on a glass dish. 5. Wash gel in water twice for 10 min with gentle rocking. Wash once again in water for a minimum of 5 min. Take a picture to document RNA quality and parameters of the gel electropho- resis run. 6. Wash the gel in 10× SSC for 10 min. 7. Wet the nylon membrane in water. Then soak both the nylon membrane and the Whatman 3MM paper in 20× SSC for 1–2 min. 8. Assemble blotting unit as shown in Fig. 1 and allow for the transfer to proceed for 12–18 h. Fig. 1 Blot unit assembly. Fill the buffer tray with 1 L 20× SSC. Place a glass or Plexiglass plate across the tray or on top of a support. Place the two lengths of presoaked filter paper over the glass or Plexiglass plate so that the ends contact the bottom of the tray. Remove any air bubbles between the sheets of filter paper and the plate by rolling a pipet several times back and forth over the surface. Position the gel upside-down on the filter paper covering the plate. Ensure that the transfer buffer moves only through the gel and not around it. Place the pre- soaked nylon membrane on top of the gel so that it covers the entire surface. Do not move the nylon membrane once it has been placed on the gel. Remove any air bubbles between the membrane and the gel by gently rolling a pipet several times back and forth over the surface. Place the three presoaked sheets of Whatman 3MM paper on top of the nylon membrane. Again, remove any air bubbles by gently rolling a pipet several times back and forth over the surface. Place a 15–20 cm stack of dry paper towels on top of the filter paper. Place a second glass or Plexiglass plate on top of the paper towels. Place the 1 kg weight on top of the plate Marc R. Reboll et al.
- 24. 9 9. After the transfer is complete, disassemble the blotting apparatus by removing the weight, paper towels, and two sheets of filter paper. Turn the gel and the nylon membrane over together, and lay them, gel side up, on a clean, dry sheet of filter paper. 10. Mark the positions of the gel lanes on the membrane using a ballpoint pen or a soft-lead pencil. Peel the gel from the mem- brane and discard it. 11. Mark the gel lanes before removing the gel from the nylon mem- brane! Without this marking, lanes will not be identifiable. 12. Fix the RNA to the blot by either baking 1 h at 80 °C in an oven or UV cross-linking. The latter method generally gives better results and enhanced sensitivity compared to baking. However, proper cross-linking requires prior optimization of the system (see Note 3). 13. If the blot will not be used immediately, it can be stored in plastic wrap at either 4 °C or room temperature for an indefi- nite period of time. The following steps vary depending on the probe labeling method, radioactive (Subheading 3.3) or nonradioactive (Subheading 3.4). However, both methods have the following general steps in com- mon: probe generation, prehybridization and hybridization with a labeled DNA-probe, removal of non-hybridized probe (washing), detection, stripping, and reprobing. Both PCR amplification of genomic regions using high molecular weight DNA or isolation of genomic fragments from pre-cloned plasmids are suitable sources for generation of probes. In general, DNA fragments should be purified by gel electrophoresis prior to the labeling reaction. The random priming technique, which is based on extension of random hexanucleotide primers hybridized to a template by simultaneous incorporation of radioactive labeled nucleotides, is the most common method for generating a radioac- tive probe from an isolated DNA fragment. The ideal size of tem- plate DNA ranges from 450 bp to 2 kb. As a general rule, a minimum of 50 bp homology is required for hybridization with target RNA in this procedure. Prepare a reaction as follows: 1. Dilute DNA to be labeled to a concentration of 2.5–25 ng in 45 μl of 10 mM Tris–HCl pH 8.0, 1 mM EDTA. 2. Denature the DNA sample by heating to 95–100 °C for 5 min. 3. Chill DNA template on ice for 5 min. 4. Centrifuge briefly to bring the contents to the bottom of the tube. 5. Start labeling reaction using Rediprime II DNA labeling kit (GE Healthcare, Pittsburg, PA). 3.3 Radioactive DNA-Probe Labeling and Detection 3.3.1 Probe Generation RNA Full-Length Characterization
- 25. 10 6. Add the template to a supplied ready-to-use reaction tube and carefully mix the components by gently flicking tube with finger. 7. Add 5 μl of [α-32 P] dCTP and carefully mix by pipetting up and down. 8. Incubate reaction at 37 °C for 10 min. 9. Stop the reaction by adding 5 μl of 0.2 M EDTA. 10. Purify the labeled probe using Illustra MicroSpin G-25 Columns (GE Healthcare, Pittsburg, PA). 11. Resuspend the resin in the supplied column by vortexing. 12. Detach the bottom closure by turning the cap. 13. Spin the column for 1 min at 735×g. 14. Place the column into a 1.5 ml tube. 15. Add labeling reaction to the center of the resin surface without disturbing the resin bed. 16. Spin for 2 min at 735×g to collect the sample at the bottom of the tube. 17. Store purified probe at −20 °C or use immediately. 1. Mix 0.58 g NaCl, 1 g dextran sulfate and 9.5 ml sterile water in 50 ml plastic tub for 10 min. Heat to 60 °C until solution turns clear, then add 0.5 ml 20 % SDS. Mix gently to avoid bubbles and incubate at 60 °C during next two steps. 2. Denaturate 1 % yeast RNA and 10 mg/ml herring sperm DNA by boiling in water bath for 5 min, then chilling on ice for 5 min. 3. Clean a hybridization tube, preheat to 60 °C, and insert blot into pre-warmed tube. 4. Add 0.5 ml herring sperm DNA and 0.5 ml RNA to the preheated hybridization buffer; mix and carefully pipet into the hybridization tube. Avoid bubbles! 5. Incubate the tube for prehybridization at 60 °C for a minimum of 30 min. 6. Radioactive probe can either be added directly to the tube or mixed with a new aliquot of hybridization buffer, which replaces the prehybridization buffer. 7. Incubate at 60 °C overnight or a minimum of 8 h. 1. Following hybridization, carefully remove the radioactive hybridization buffer by pipetting or draining from tube. Buffer can be directly disposed into radioactive waste or stored at −20 °C for a maximum of 1 week and used once more for hybridization (see Note 4). 3.3.2 Prehybridization and Hybridization with a Labeled DNA-Probe 3.3.3 Washing Marc R. Reboll et al.
- 26. 11 2. Wash the blot in the tube with Buffer A for 2 min to remove residual, unbound radioactive probe. 3. Place blot in a glass dish and wash once again with Buffer A for 5 min with gentle agitation. Check counts using a hand coun- ter and use this initial count as a baseline value of 100 % to estimate the efficiency of next wash steps. 4. Wash blot in Buffer B at room temperature for 15 min with gentle rocking. Check counts every 5 min. If the counts are reduced to 3 %, skip the next step. 5. Wash in Buffer C for 15 min at 62 °C. Check counts every 5 min. If the counts are lower than 10 %, stop the wash. 6. Wash in Buffer D for 5 min at room temperature. 1. Wrap blot in plastic wrap and expose to X-ray film. Typically, several hybridization experiments using different labeled genomic probes are required to characterize all transcripts from a single gene and provide visualization of an appropriate positive control. Therefore, a single blot is usually probed several times. However, prior to rehybridizing with new probes, the old label should be carefully removed from the blot. This procedure is called stripping. 1. First incubate the membrane in 0.1 % SDS at 95–100 °C for 10–20 min. 2. Next, wash the membrane in 2× SSC for 5 min at room tem- perature and start the next round of hybridization with a new probe. Expose blot to X-ray film to ensure that radioactive probe is completely removed. 3. Begin new hybridization. The digoxigenin (DIG) system—based on the steroid hapten, digoxigenin, which occurs in certain digitalis plants—is an effective method for nonradioactive labeling and detection of nucleic acids [4]. Digoxigenin is suitable for detection purposes based on three char- acteristics. First, high affinity antibodies can be easily generated if digoxigenin is coupled to a suitable carrier molecule. Second, there are no endogenous background problems with the anti-digoxigenin antibodies as in the case of other haptens, such as biotin, because digoxigenin occurs exclusively in digitalis plants. Third, digoxigenin can be coupled to nucleotides like dUTP and then incorporated into nucleic acids using Klenow, Taq, or RNA polymerases to generate DIG-labeled probes. These probes can be used in stan- dard blotting and hybridization procedures and detected with anti-digoxigenin conjugates such as fluorescent or alkaline-phos- phatase labeled antibodies [5]. 3.3.4 Detection 3.3.5 Stripping and Reprobing 3.4 Nonradioactive DNA-Probe Labeling and Detection RNA Full-Length Characterization
- 27. 12 The nonradioactive DNA probe is labeled incorporating DIG- labeled dUTP by PCR using the PCR DIG Probe Synthesis kit and Anti-DIG-AP antibody for detection (see Subheading 2). This method is particularly recommended when template is available in limited quantity, is only partially purified, or is very short [6, 7]. The PCR DIG Probe Synthesis kit is especially designed for gen- eration of highly sensitive hybridization probes suitable for detec- tion of low (single) copy sequences. 1. In a 1.5 ml tube, prepare a DIG-labeled DNA probe that is going to be used as hybridization probe adding the reagents in the amounts shown in Table 1. Note that for DIG-labeled probes <1 kb long, a 1:3 DIG-dUTP–dTTP ratio (standard ratio) is recommended. However, for longer DIG-labeled probes (i.e., >1 kb long), a 1:6 DIG-dUTP–dTTP ratio should be used to avoid low yield of DIG-labeled PCR product. 2. In another 1.5 ml tube, prepare the unlabeled positive control adding the reagents and amounts according to Table 1. Mix the reagents and centrifuge briefly. 3. Transfer samples to 0.2 or 0.5 ml tubes suitable for PCR and mix thoroughly. 4. Place samples in thermocycler and begin cycling program. The optimal cycling conditions depend on the combination of template, primers, and thermocycler (see Note 5). The follow- ing conditions are a good starting point: 3.4.1 Probe Generation Table 1 DIG-probe labeling by PCR using PCR DIG Probe Synthesis kit Reagent DIG-labeled probe Un-labeled controla Final concentration Sterile double-distilled water Variable volume Variable volume – 10× PCR buffer with MgCl2 (vial 3) 5 μl 3 μl 1× 10× PCR DIG mix (vial 2) 5 μl – 200 μM 10× dNTP stock solution (vial 4) b 3 μl 200 μM Forward and reverse primers [50 μM] 0.5 μl 0.3 μl 0.5 μM each primer Enzyme mix (Expand High Fidelity, vial 1) 0.75 μl 0.45 μl 2.6 units total enzyme Template DNA Variable volume Variable volume 10 ng genomic DNA or 10 pg plasmid DNA Final volume 50 μl 30 μla 30 or 50 μl a The use of a lower total reaction volume for the un-labeled control than the DIG-labeled probe is only to save reagents of the PCR DIG Probe Synthesis kit (Roche Applied Science, Indianapolis, IN) b For DIG-labeled probes<1 kb long: use 1:3 DIG-dUTP–dTTP ratio and do not add 10× dNTP stock solution (vial 4). For DIG-labeled probes>1 kb long use 1:6 DIG-dUTP–dTTP ratio and add 5 μl of 10× dNTP stock solution (vial 4) Marc R. Reboll et al.
- 28. 13 (a) Initial denaturation step (before the first cycle): 95 °C for 2 min (b) PCR amplification (35 cycles): 95 °C for 30 s; 60 °C for 30 s; 72 °C for 40 s (c) Final elongation step: 72 °C for 7 min The labeling efficiency for PCR-labeled probes can be quickly estimated by gel electrophoresis as described below: 5. Prepare a 1–1.5 % w/v agarose gel in TAE buffer and add 10 μl SYBR® Safe DNA Gel Stain 10,000× concentrate (Invitrogen, Carlsbad, CA) per ml of agarose solution. Alternatively, gels can be stained with ethidium bromide, as described above. 6. Load 5 μl of each PCR amplification product on agarose gel. Include a DNA molecular weight marker. Electrophorese the samples at 60 V for 40 min or until the DIG-labeled and unla- beled probes are adequately separated. DIG-labeled probes are larger than unlabeled probes; thus, these probes migrate more slowly compared to unlabeled DNA of the same size. The inten- sity of the stained DIG-labeled probe should be equal to, or slightly less than, the intensity of the unlabeled probe DNA. If these conditions are met, then the DIG-probe was likely labeled efficiently and the standard amount of labeled probe (2 μl of PCR product per ml hybridization buffer) can be used in hybrid- ization reactions. If the intensity of the labeled PCR product band is very strong on the gel, 0.5 μl probe per hybridization buffer should be used (see Note 6). If the signal is very faint, up to 4 μl probe per ml hybridization buffer should be used [5]. 1. After fixing the RNA to a Zeta Probe membrane, or any similar positively charged nylon membrane, incubate the membrane in 2× SSC buffer to remove any agarose or salts remaining from the RNA capillary transfer. First incubate the membrane in 0.1 % SDS at 95–100 °C for 10–20 min. 2. Place membrane on 3MM paper with RNA face up. 3. For prehybridization and hybridization, use ready-to-use DIG Easy Hyb solution. In a hybridization oven at 50 °C, pre-warm a 50 ml tube containing 16 ml of DIG Easy Hyb solution. 4. Pre-warm an empty hybridization tube in the hybridization oven at 50 °C. 5. Prehybridization step: insert membrane into the pre-warmed hybridization tube and immediately add 10 ml of the pre-warmed DIG Easy Hyb solution prepared in step 3 (see Notes 7 and 8). Incubate with rotation in a hybridization oven at 50 °C for at least 30 min. 6. In a 1.5 ml tube, mix 200 μl Easy Hyb solution with the DIG- labeled DNA probe (2 μl DNA probe labeled by PCR per ml of DIG Easy Hyb solution) (see Note 6). 3.4.2 Prehybridization and Hybridization with a DIG-Labeled DNA-Probe RNA Full-Length Characterization
- 29. 14 7. Denature probe by boiling 10 min at 100 °C, then immediately quenching on ice. 8. Add the denatured probe to the remaining 6 ml of the pre- warmed DIG Easy Hyb solution prepared in step 3 to obtain 6 ml of hybridization solution. If a previous hybridization solu- tion (containing probe from a previous northern blot experi- ment) is going to be used, incubate it at 68 °C for 10 min just before adding it to the hybridization tube with the membrane. 9. Hybridization step: immediately after the 30 min prehybridiza- tion step (step 11), replace the prehybridization solution in the hybridization tube with the 6 ml hybridization solution prepared in step 8. Incubate, rotating slowly, at 50 °C over- night (16–18 h). 1. After the hybridization is complete, save the hybridization solution at −20 °C. 2. Keep membrane in the hybridization tube and wash twice with 100 ml Low Stringency Buffer (high salt concentration and low temperature) at room temperature for 5 min each time. 3. Preheat High Stringency Buffer (low salt concentration and high temperature) at 50 °C. 4. Remove used Low Stringency Buffer and immediately add preheated High Stringency Buffer. 5. Wash with 100 ml High Stringency Buffer at 50 °C twice for 15 min each time. Probe–target hybrids are detected with an enzyme-linked immu- noassay, which may be more sensitive than radioactive detection procedures when the optimal experimental conditions are followed [4, 5]. Prepare the following solutions using DIG Wash and Block Buffer kit just before starting the detection: ● 500 ml 1× washing buffer: 50 ml 10× Washing Buffer (shake vigorously before use)+450 ml double-distilled RNAse-free water. ● 50 ml 1× maleic acid buffer: 5 ml 10× Maleic Acid Buffer+45 ml double-distilled RNAse-free water. ● 20 ml 1× blocking solution: 2 ml 10× Blocking Solution+18 ml 1× Maleic Acid buffer. ● 20 ml 1× detection buffer: 2 ml 10× Detection Buffer+18 ml double-distilled RNAse-free water. 1. Equilibrate the membrane in a clean tray (treated with anti- RNasespray)containing50ml1×WashingBufferfor1–5min. 2. First wash the hybridization tube with soap and water, then treat it with RNAse Zap (Applied Biosystems, Carlsbad, CA) and rinse with RNAse-free water. 3.4.3 Washes 3.4.4 Detection Marc R. Reboll et al.
- 30. 15 3. Place the membrane into hybridization tube and add 10 ml 1× Blocking Solution. Incubate for 30 min in hybridization oven at room temperature. 4. Prior to each use, centrifuge the anti-Digoxigenin-AP in the original vial, at 10,000×g for 5 min. Prepare the antibody solution adding 0.5 μl anti-Digoxigenin-AP taken carefully from the surface to 10 ml Blocking Solution to obtain the anti- body solution (dilution 1:20,000) (see Note 9). 5. Remove Blocking Solution from hybridization tube and add 10 ml of antibody solution prepared in step 5 of Subheading 3.3.3. Incubate for 30 min at room temperature in the hybridization oven with rotation. In a 0.2 ml tube, combine up to 5 μg total RNA, 1 μl primer (see Note 4), 1 μl 10 mM dNTP mix, and DEPC-treated water to a final volume of 10 μl. Make sure to include a negative control as well as a −RT control. 6. Transfer the membrane to a clean, RNase-free tray, and add 100 ml 1× Washing Buffer. Incubate for 15 min with shaking. 7. Repeat step 6. 8. Pour off washing buffer and add 50 ml 1× Detection Buffer (DIG block and wash buffer kit). Incubate at room tempera- ture for 5 min. 9. Prepare substrate solution: add 10 μl CDP-star to 1 ml Detection Buffer and mix thoroughly. 10. Place the membrane with RNA-side facing up on a clean ace- tate sheet (see Note 10). Cover the membrane with 1–2 ml of substrate solution. 11. Immediately cover the membrane with a second acetate sheet to spread the substrate evenly over the membrane, avoiding air bubbles. Incubate for 5 min at room temperature. 12. Squeeze out excess liquid and seal edges of the development folder. Do not let the membrane dry completely. 13. Expose sealed envelope containing the membrane to one of the following at room temperature: (a) Lumi-Imager F1 Workstation (5–20 min). The Lumi- Imager allows for rapid, quantitative analysis of the chemi- luminescent signal without X-ray film. (b) KODAK BioMax XAR film or Lumi-Film X-ray film (1–20 min) (see Note 11). Adjust the exposure time to get a darker or lighter band pattern, depending on the results. Table 2 shows a summary of the most common problems that might arise during DIG labeling and detection and how to solve or prevent them. RNA Full-Length Characterization
- 32. 17 We recommend the blot be stripped soon after detection as follows: 1. Heat 0.1 % SDS solution to 100 °C. 2. Transfer the membrane into a clean, RNAse-free tray and cover immediately with plenty of the preheated 0.1 % SDS solution. Incubate for 10–60 min at 100 °C. 3. Wash the membrane with 100 ml 2× SSC for 5 min with shaking at room temperature. 4. Store stripped blot wet in Maleic acid Buffer at 4 °C. 4 Notes 1. Make sure that there are no air bubbles in the gel or trapped between the wells which could possibly connect single wells or lead to inconsistent sample runs. Air bubbles can be carefully removed with a plastic pipette tip before the gel sets. 2. The 18S and 28S ribosomal RNA bands should appear as sharp bands. If the ribosomal bands in a given lane are not sharp, but appear as a smear towards smaller-sized RNAs, it is likely that the RNA sample suffered major degradation during prepara- tion. The 28S ribosomal RNA band should be present at approximately twice the intensity of the 18S rRNA band. As the 28S rRNA is more labile than the 18S rRNA, equal intensities of the two bands generally indicates that some deg- radation has occurred. 3. UV light can damage the eyes and skin. Always wear suitable eye and face protection. 4. Pre-used hybridization buffer should be denatured at 95 °C for 5 min before use. 5. To obtain a high yield of DIG-labeled PCR product, always optimize the PCR parameters (cycling conditions and concentrations of template, MgCl2, and primers) for each tem- plate and primer set in the absence of DIG-dUTP before attempting incorporation of DIG. 6. An excessive probe concentration in the hybridization solution causes cloudy hybridization background. If this happens, reduce probe concentration to 0.5–1 μl per ml DIG Easy Hyb buffer. 7. Use enough buffer to completely cover the membrane during prehybridization and hybridization incubations. The amount needed will depend on the shape and capacity of the container used for the incubations. Uneven distribution of probe during hybridization produces an irregular and cloudy background and it is caused by using too little hybridization solution. 3.4.5 Stripping and Reprobing RNA Full-Length Characterization
- 33. 18 Use at least 3.5 ml of hybridization solution per 100 cm2 of membrane. If roller bottles (hybridization tubes) are used for incubation, add at least 6 ml hybridization solution per bottle. 8. Do not allow the membrane to dry at any time from the beginning of prehybridization through the final detection. If the membrane dries or sticks to a second membrane, the assay will have a high background. 9. Several uses and centrifugation steps of the anti-digoxigenin- AP conjugate can cause a certain loss of material, which must be compensated by use of larger amounts. 10. Do not use plastic wrap to cover the membrane during the detection step: use hybridization bags, acetate sheet protec- tors, or two sheets of transparency film. 11. Luminescence continues for at least 24 h and signal intensity remains almost constant during the first hours. Multiple expo- sures at different times can be taken to achieve the desired signal strength. References 1. Holtke HJ, Ankenbauer W, Muhlegger K, Rein R, Sagner G, Seibl R et al (1995) The digoxi- genin (DIG) system for nonradioactive labeling and detection of nucleic acids—an overview. Cell Mol Biol 41:883–905 2. Rueger B, Thalhammer J, Obermaier I, Gruenewald-Janho S (1997) Experimental pro- cedure for the detection of a rare human mRNA with the DIG System. Front Biosci 2:C1–C5 3. Sambrook J, Russell D (2001) Molecular clon- ing: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 4. Hloch P, Hoffmann K, Kruchen B, Rueger B (2001) The DIG system—a high sensitive substitute of radioactivity in northern blot analysis. Biochemica 2:24–25 5. Roche Applied Science (2003) DIG application manual for filter hybridization, 3rd edn, Roche Diagnostics GmbH, Roche Applied Science, Mannheim, Germany. http://lifescience.roche. com/wcsstore/RASCatalogAssetStore/ Articles/05353149001_08.08.pdf 6.Rost A, Kohler T, Heilmann S, Lehmann J, Remke H, Rotzsch W (1995) A rapid and sim- ple method to prepare digoxigenin-labeled DNA-probes by using PCR-generated DNA- fragments. Eur J Clin Chem Clin Biochem 33:A59 7. Finckh U, Lingenfelter PA, Myerson D (1991) Producing single-stranded DNA probes with the Taq DNA polymerase: a high yield protocol. Biotechniques 10:35–39 Marc R. Reboll et al.
- 34. 19 M. Lucrecia Alvarez and Mahtab Nourbakhsh (eds.), RNA Mapping: Methods and Protocols, Methods in Molecular Biology, vol. 1182, DOI 10.1007/978-1-4939-1062-5_2, © Springer Science+Business Media New York 2014 Chapter 2 Rapid Mapping of RNA 3′ and 5′ Ends Victoria Zismann and Mahtab Nourbakhsh Abstract In recent years, an enormous progress has been made in applied genomics leading to identification and isolation of novel cDNAs. However, most attempts result in the acquisition of transcribed sequences that represent only a part of the mRNA’s complete sequence. Rapid Amplification of cDNA Ends (RACE) is a technique used in molecular biology to obtain the full length sequence of an RNA transcript found within a cell. Since the first report of this technique, many significant improvements have been made on the basic approach. This chapter describes the most recent update of the relatively simple and versatile classic RACE protocol. Key words Gene expression, mRNA mapping, Lambda cDNA library, cDNA end amplification, RACE amplification 1 Introduction Rapid Amplification of cDNA Ends (RACE) is used to extend partial cDNAs by amplifying the 5′ or 3′ sequences of the corre- sponding mRNAs using gene-specific primers [1, 2]. The tech- nique requires knowledge of only a small region of the sequence within the known partial cDNA (Fig. 1a). The first step begins with cDNA synthesis using cellular RNA. During this step, the Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV RT) utilizes terminal transferase activity to add three to five resi- dues to the 3′ end of the polymerized cDNA upon reaching the end of an RNA template. An additional primer, which contains a terminal stretch of annealing residues, is added to the reaction and serves as an extended template for reverse transcription. MMLV RT switches templates from the mRNA molecule to the primer generating a complete cDNA copy of the original RNA with the additional end sequence. This step is called Switching Mechanism at 5′ End of RNA Template (SMART). The relationship of the primers used in the SMART RACE reactions to the template and resulting RACE products is shown in detail in Fig. 1a.
- 35. 20 A straightforward alternative approach to RACE relies on amplification of cDNA ends from commercially available cDNA libraries. The cDNA libraries represent the total RNA from a cell line or tissue integrated into lambda vectors as single copies. This strategy takes advantage of both stability and integrity of pre-made cDNA libraries. Commercially available cDNA libraries are designed and evaluated to preserve highest independent copy numbers of full-length cDNAs. In addition, the 3′ and 5′ adjunct arms of the vector allow for more stringent design for 3′ or 5′ amplification primers (Fig. 1a). 2 Materials 1. SMART RACE cDNA Amplification (Clontech, Inc; Mountain View, CA). 2. Advantage PCR Kit and Polymerase Mix (Clontech). 3. PCR clean-up Gel extraction NucleoTraPCR or NucleoTrap (Clontech). 4. February. 5. Thermocycler (e.g., Applied Biosystems, Inc). 2.1 Rapid Amplification of cDNA Ends (RACE) Fig. 1 The relationship of gene-specific primers to the cDNA template by PCR- based amplification of cDNA ends. (a) This diagram shows a generalized first- strand cDNA template and direction and position of gene-specific primers, GSP1 and GSP2, required for Rapid Amplification of cDNA Ends (RACE). (b) This dia- gram shows direction and position of gene-specific primers, GS5′P and GS3′P, and lambda-specific primers, V5′P and V3′P, for identification of cDNA ends using premade cDNA libraries Victoria Zismann and Mahtab Nourbakhsh
- 36. 21 1. Storage medium: 100 mM NaCl, 50 mM Tris–Cl, pH 7.5 (25 °C), 10 mM MgSO4, 2 % (w/v) gelatin, 7 % (w/v) DMSO. 2. 10× PCR buffer: 500 mM Tris–Cl, pH 9.2 (25 °C), 160 mM (NH4)2SO4, 22.5 mM MgCl2. 3 Methods The following reaction is capable of converting 0.1–1 μg of poly(A+) RNA into first-strand cDNA. The use of poly(A+) RNA is recommended if there is an evidence for polyadenylation, such as poly(A) signal in genomic sequence or poly(A) tail at cDNA 3′ end. The use of total RNA may increase background noise and should only be considered if the target RNA is not polyadenylated. 1. Combine appropriate amount of poly(A+) RNA with 1 μl of a 12 μM 5′-antisense or 3′-sense cDNA primer, and 1 μl of SMART II A oligo included in the SMART RACE cDNA Amplification kit (Clontech). The appropriate amount of poly(A+) RNA strongly depends on the specific abundance of a transcript and should be determined using different amounts of poly(A+) RNA in parallel reactions. 2. Add RNase-free water to a final volume of 5.0 μl for each reaction. 3. Mix contents and spin tubes briefly in a microcentrifuge. 4. Incubate the tubes at 70 °C for 2 min. 5. Cool the tubes on ice for 2 min. 6. Spin briefly to collect contents to bottom of tubes. 7. Add 2 μl 5× First-Strand Buffer (SMART RACE cDNA Amplification Kit) and 1 μl MMLV Reverse Transcriptase to a total volume of 10 μl. 8. Mix reaction mixture gently by pipetting. 9. Spin the tubes briefly. 10. Incubate the tubes at 42 °C for 1.5 h. 11. Heat reaction at 72 °C for 10 min and store at −20 °C. The reaction contains extended 3′ or 5′ cDNA sequences. Only a fraction of this material should be used for amplification of the cDNA end of interest (see Note 1). 12. Mix the following reagents for each PCR reaction: (a) 34.5 μl PCR-Grade Water (b) 5 μl 10× Advantage 2 PCR Buffer (c) 1 μl dNTP Mix (10 mM) (d) 1 μl 50× Advantage 2 Polymerase Mix 2.2 Identification of cDNA Ends Using Premade Lambda cDNA Libraries 3.1 Rapid Amplification of cDNA Ends (RACE) Mapping RNA Ends
- 37. 22 13. Mix well by vortexing (without introducing bubbles), and briefly spin in a microcentrifuge. For 5′-RACE add: (a) 2.5 μl 5′-RACE-Ready cDNA (b) 5 μl Universal Primer A Mix (c) 1 μl gene-specific 5′ primer GSP2 (10 μM) For 3′-RACE add: (a) 2.5 μl 3′-RACE-Ready cDNA (b) 5 μl Universal Primer A Mix (c) 1 μl gene-specific 3′ primer GSP2 (10 μM) 14. Perform 20–25 cycles using the following program (see Note 2) (a) 94 °C for 30 s (b) 78 °C for 30 s (c) 72 °C for 3 min (see Note 3) 15. Prepare a 1–1.5 % w/v agarose gel in TAE buffer and ethidium bromide (EtBr) to a final concentration of approximately 0.2–0.5 μg/ml (see Note 4). 16. Load 5 μl of each PCR amplification product on the gel; include a DNA molecular weight marker for size estimation, and begin electrophoresis at 60 V for 40 min or until the amplified fragment and unincorporated primers are clearly separated. 17. Locate the position of your fragment under UV light. If you find no or too many fragments, adjust the PCR conditions as outlined in Table 1. 18. Excise the DNA fragment of interest using a clean scalpel and transfer it to a clean 1.5-ml microcentrifuge tube (see Note 5). 19. For every 100 mg of agarose, add 300 μl of Buffer NE (NucleoTraPCR) and vortex the NucleoTrap Suspension thor- oughly until the beads are completely resuspended. 20. Add 10 μl of NucleoTrap Suspension or more (4 μl of NucleoTrap Suspension for each 1 μg of DNA) and incubate the sample at 50 °C for 5–15 min. Vortex briefly several times during the incubation period. 21. Centrifuge the sample at 10,000×g for 30 s at room tempera- ture; discard supernatant. 22. Add 500 μl of Buffer NT2 to the pellet. Vortex briefly and centrifuge at 10,000×g for 30 s at room temperature. Remove supernatant completely, and repeat steps 20 and 21. 23. Add 500 μl of Buffer NT3 to the sample. Vortex briefly and centrifuge the sample at 10,000×g for 30 s at room tempera- ture. Remove the supernatant completely and repeat this step. Victoria Zismann and Mahtab Nourbakhsh
- 38. 23 24. Air-dry DNA pellet for 10–15 min. 25. Add 20–50 μl of Buffer NE and resuspend the pellet by vortexing. 26. Elute DNA by incubating the sample at room temperature for 10–15 min. 27. Centrifuge the sample at 10,000×g for 30 s at room tempera- ture, and then transfer the supernatant containing the purified DNA to a fresh tube. The isolated fragment(s) can now be directly cloned into a T/A-type PCR cloning vector (e.g., Life Technologies). This approach relies on amplification of a cDNA sequences from commercially available human cDNA libraries which represent the total RNA from a cell line or tissue. The main advantage of premade libraries is that they contain high-quality, full-length cDNAs inserted into a self-replicating lambda vector. Once the sequence is available in the form of a cDNA library, individual processed segments of the original cDNA can be isolated and examined with relative ease. 3.2 Identification of cDNA Ends Using Premade cDNA Libraries Table 1 Troubleshooting guide for SMART RACE cDNA Amplification kita Problem Recommendations 5′ or 3′-RACE product is not the expected size or is absent The cause may be a GCrich template. Use Clontech Mixes for efficient amplification of GC-rich templates. PCR parameters may need to be optimized for these templates No amplified 3′ or 5′ products after the minimum number of cycles at 68 °C Return tube(s) to the thermal cycler and run five additional cycles. If the product still does not appear, add an additional three to five cycles at 68 °C. If you are still unsuccessful, run a new PCR experiment, changing the annealing temperature in the third set of cycles from 68 °C to 65 °C. This last program is especially useful if Tm close to 70 °C Neither 3′ nor 5′ amplified products Check the quality of first-strand cDNA (if generated from poly A+RNA) using a 32P-labeling procedure. Repeat the first-strand synthesis, substituting 1 μl of 0.1 μCi/μl [α-32P] dATP or dCTP for 1 μl of water. Run the reaction products on an alkaline agarose gel, and examine the banding pattern by autoradiography. If the first-strand reaction was successful, you should see a banding pattern similar to that produced by your RNA. Mammalian poly A+RNA typically produces a smear from 0.5 to 12 kb. Mammalian total RNA usually exhibits two bright bands at 1.9 kb and 4.5 kb Multiple 5′- and/ or 3′-RACE products By multiple fragments you can generally start with the largest fragment from each RACE reaction, because it is most likely to be a true, complete RACE product. However, in the long run you should try to eliminate nonspecific fragments by troubleshooting the reactions a The information in this table was partially taken and modified from SMART RACE cDNA Amplification kit manual from Clontech; Mountain View, CA Mapping RNA Ends
- 39. 24 For this method gene-specific and lambda vector-specific primers are utilized. The relationship of the required primers is shown in detail in Fig. 1b. In general, primers should be: ● 23–28 nt ● 50–70 % GC ● Tm≥65 °C; the best results are obtained when Tm>70 °C For amplification of 5′ or 3′ ends, lambda vector-specific prim- ers should hybridize 50 nt upstream or downstream to the cDNA cloning site, respectively. 1. Combine 107 plaque forming units of a λ cDNA library in 1–5 μl of storage medium. 2. Add 4.5 μl of 10× PCR buffer. 3. Add 5 μl of 10 mM dNTPs and 50 pmol of each lambda (V5′P or V3′P) and gene-specific primer (GS5′P or GS3′P). 4. Add sterile water to a final volume of 45 μl. 5. Incubate reaction at 95 °C for 10 min in a thermocycler to denature phage particles. 6. Incubate reaction 5 min at 75 °C. Within this step, add 5 μl of a pre-made master polymerase mix: 0.5 μl 10× PCR buffer, 0.5 μl polymerase, and 4 μl sterile water. 7. Continue with standard PCR reaction cycles. For example, 30 cycles of denaturation at 94 °C for 45 s, annealing at 63 °C for 30 s, and extension at 72 °C for 3 min. 8. Continue with the steps 15–17 of Subheading 3.1 for detec- tion of cDNA fragment of interest. 9. If you find no or too many cDNA fragments, adjust the PCR conditions as outlined in Table 2. 10. Continue with the steps 18–27 of Subheading 3.1 for cloning and characterization of cDNA fragment of interest. 4 Notes 1. Prepare enough PCR Master Mix for all PCR reactions, plus one extra reaction, to ensure sufficient volume. The same Master Mix can be used for both 5′- and 3′-RACE reactions. 2. Because the necessary number of cycles depends on the abun- dance of the transcript, you may need to determine the optimal cycling parameters for your gene empirically. 3. If fragments >3 kb are expected, add 1 min for each additional 1 kb. Victoria Zismann and Mahtab Nourbakhsh
- 40. 25 4. Prepare and use a 1,000-fold master EtBr solution (0.2–0.5 mg/ ml) and 1 μl/ml agarose gel volume. 5. EtBr solution must be handled with extreme caution and decontaminated prior to disposal. Caution is required by han- dling with UV which can cause serious damage to your eyes and skin. References Table 2 Troubleshooting guide for cDNA end amplification using cDNA librariesa Problem Recommendations 5′ or 3′ RACE product is not the expected size or is absent The cause may be a GC-rich template. Use buffer conditions for efficient amplification of GC-rich templates. PCR parameters may need to be optimized for these templates Depending on cDNA abundance or the quality of the cDNA library you may use up to 108 plaque forming units No amplified 3′ or 5′ products after the minimum number of cycles at 68 °C Change the annealing temperature in the third set of cycles from 72 °C to 68 °C. This last program is especially useful if Tm close to 70 °C Neither 3′ nor 5′ amplified products Check the quality of cDNA library using primers for abundant cDNAs Multiple 5′- and/or 3′-RACE products By multiple fragments you can generally start with the largest fragment from each PCR reaction. If you cannot isolate a single band, you should try to eliminate nonspecific fragments by troubleshooting the reactions by changing the annealing temperature, reducing cycle number, or using high stringent buffer conditions a Although most convenient, the use of cDNA libraries in PCR reactions might cause artifacts based on high copy num- ber of high abundant cDNAs in a library. Thus, you may require optimizing the PCR conditions step by step 1. Yeku O, Frohman MA (2011) Rapid amplifica- tion of cDNA ends (RACE). Methods Mol Biol 703: 107–122 2. Frohman MA, Dush MK, Martin GR (1988) Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc Natl Acad Sci U S A 85: 8998–9002 Mapping RNA Ends
- 41. 27 M. Lucrecia Alvarez and Mahtab Nourbakhsh (eds.), RNA Mapping: Methods and Protocols, Methods in Molecular Biology, vol. 1182, DOI 10.1007/978-1-4939-1062-5_3, © Springer Science+Business Media New York 2014 Chapter 3 Single Nucleotide Mapping of RNA 5′ and 3′ Ends Mahtab Nourbakhsh Abstract Nuclease protection assay is a sensitive method for detection, quantitation, and mapping of a specific RNA in an extremely heterogeneous mixture of RNAs, such as total cellular RNA. The assay is based on a small volume solution hybridization of a single-stranded synthetic antisense and labeled RNA probe to a RNA sample. Thus, it is much more efficient than the common immobilized hybridization on a membrane, such as in northern-blot analysis. After solution hybridization, different nucleases are used to remove any remaining single-stranded nucleotides within the probe and sample RNA by digestion. Then, the remaining probe-target hybrids are purified and separated on a denaturing polyacrylamide gel. Using a radioactive labeled probe, the protected probe can be visualized by direct autoradiography and the copy number can be calculated based on the specific radioactivity of the RNA probe and the length of protected fragment. Because of its high sensitivity and resolution, nuclease protection assay is the most effective procedure for mapping internal and external boundaries in mRNA compared to other RNA detection methods such as RT-PCR. Key words Intron, 5′ UTR, 3′ UTR, Double stranded RNA, Hybridization, Nuclease, S1 RNA mapping 1 Introduction Molecular characterization of genomic sequences involves the qualitative and quantitative analysis of possibly transcribed sequences. Transcribed regions can be hypothetically determined by aligning Expressed Sequence Tags (ESTs) with genome sequences [1]. However, available ESTs databases represent only a fraction of transcribed regions. This makes further experimental analysis indispensable. The current book includes three most frequently used technical procedures for mapping, detecting, and quantifying a particular RNA in a total RNA sample: Northern blot analysis, nuclease pro- tection assays, and reverse transcription-polymerase chain reaction (RT-PCR). Although each of these techniques can be used for qualitative and quantitative RNA detection, each procedure has decisive advantages and/or limitations. Northern-blot analysis is
- 42. 28 the only method that can provide information about the size of the mature transcripts. RT-PCR is the most convenient, fast, and sensitive method for comparative detection and estimation of rel- ative abundance of transcripts in different samples. The principle of nuclease protection assays is based on hybridization of a labeled synthetic antisense RNA probes to a target RNA which forms a double-stranded RNA fragment protected against specific single strand nucleases (see Fig. 1). Following nuclease treatment and removing of single-stranded sequences, the length and the copy number of protected probe fragments correspond to the bound- aries and number of target RNA in a sample. As truncation of the full-length probe is the indicative step, it is crucial to use a probe which overspans the possible homology region. In most cases, probes are designed to convey unrelated vector sequences (see Fig. 1). The potential use of different antisense probes in nuclease pro- tection experiments allows for simultaneous quantification of dif- ferent RNA species in a single sample [2]. Thus, several different target RNAs or several different fragments of a single target RNA can be simultaneously detected in samples. First, this approach can Fig. 1 Nuclease protection assay principle.The method is based on hybridization of a labeled synthetic antisense RNA probes to a target RNA which forms a dou- ble-stranded RNA fragment. S1 or similar nucleases remove single-stranded sequences. The remaining fragment signal corresponds to the length and the copy number of a target RNA. Digestion of the full-length probe is an indicative step. Therefore, unspecific vector sequences should be co-transcribed which overspans a possible homology region Mahtab Nourbakhsh
- 43. 29 discriminate between closely related targets if probes are designed to span the regions where the related genes differ at the most. Second, probes can be designed to protect fragments of different sizes of target RNAs. Nuclease protection assay is thereby suitable for precise single nucleotide mapping of external and internal junc- tions in RNA including transcription initiation or termination sites as well as intron and exon boundaries. This method is an important tool for analysis of alternative splicing [3]. Although DNA can serve as a probe as well, RNA probes are mostly preferred because RNA–RNA duplexes are more stable than RNA–DNA duplexes in solution hybridization. The antisense RNA probe is usually synthesized using available in vitro Transcription kits and radioactive nucleotides. This step requires a DNA template containing antisense sequence downstream to a synthetic promoter, such as T7. Although end-labeling of RNA probe is feasible as well, incorporation of numerous radioactive nucleotides during transcription reaction reveals a probe with sig- nificantly higher specific activity. Using the MAXIscript Kit described in this chapter, radiolabeled RNA probes can be synthe- sized in a 10 min reaction. The Kit can be used to incorporate any labeled nucleotide into RNA using Sp6, T3 or T7 polymerases. Following in solution hybridization of RNA target and anti- sense probe a nuclease treatment removes all single-stranded sequences, precisely at the nucleotide 3′ and 5′ to double stranded sequence. The last stage of the assay involves the separation of pro- tected probe and RNA fragments in a denaturing gel. A short gel run (15 cm) is sufficient for most quantification purposes. For pre- cise mapping experiments and exact determination of the size of the protected fragments, they may be resolved on a denaturing sequencing gel (60 cm) in combination with a RNA marker or “sequencing ladder” reaction [4]. This chapter provides the complete RNase protection assay protocol including support protocols for synthesis of labeled probes, and quantitation and mapping of target mRNA. 2 Materials 1. MAXIscript® kit (Life Technologies). 2. DNA template (see Note 1). 3. Labeled nucleotide (see Note 2). 4. Trichloroacetic acid: molecular biology grade. 5. Ethanol: ACS reagent grade. 6. 0.5 M EDTA. 7. Centri-Spin™ 40 Columns (Life Technologies). 2.1 Synthesis of RNA Antisense Probe Single Nucleotide RNA Mapping
- 44. 30 1. RPA III™ ribonuclease protection assay kit (Life Technologies). 2. Heat block (42–45 °C and 85–95 °C). 3. RNAse-free polypropylene microfuge tubes and pipette tips. 4. Microcentrifuge (10,000×g). 5. 100 % ethanol (ACS grade). 6. Trichloroacetic acid (molecular biology grade). 1. Urea (high quality). 2. 40 % Acrylamide (acryl–bis-acryl=19:1). 3. 10× TBE (0.9 M Tris base, 0.9 M Boric Acid, 20 mM 0.5 M EDTA). 4. 10 % ammonium persulfate. 5. EMED. 6. Vertical S2 Gel Electrophoresis Apparatus Life Technologies. 7. Power supply. 8. Gel Dryer Model 583 Bio-Rad. 3 Methods 1. Thaw the frozen reagents, mix, and microfuge briefly to pre- vent loss and/or contamination of material by opening the lid. 2. Keep all reagents on ice except the 10× Transcription Buffer. 3. Vortex the 10× Transcription Buffer several times at room temperature until it is completely in solution (see Note 3). 4. Assemble transcription reaction according to manufacturer’s recommendations at room temperature by adding the DNA, water, nucleotides, and 10× Transcription Buffer (see Note 4). 5. Mix thoroughly by pipetting the mixture up and down gently. 6. Incubate the reaction for 10 min to 1 h at 37 °C (see Note 5). 7. Add 1 μl TURBO DNase, mix well, and incubate at 37 °C for 15 min (see Note 6). 8. Add 1 μl of 0.5 M EDTA to stop the reaction to inactivate DNase and block the heat-induced RNA degradation. 9. Purify the transcripts using Centri-Spin™ 40 Columns (see Note 7). The amount of RNA probe required will depend on the abun- dance of the mRNA being detected and on the specific activity of the probe. 5–20 μg of total RNA is sufficient for most purposes. It is important to set up the hybridization with threefold to tenfold molar excess of the probe over the target mRNA. 2.2 Probe-Target Hybridization and RNAse Digestion 2.3 Denaturing Acrylamide Gel 3.1 Synthesis of RNA Antisense Probe 3.2 Hybridization and RNase Digestion Mahtab Nourbakhsh
- 45. 31 Further detailed guidelines for optimizing amounts of probe and sample RNA are given in manufacturer’s protocol. 1. For each experimental tube, mix about 150–600 pg (2–8×104 cpm) of RNA antisense probe per 10 μg total sam- ple RNA (0.6 μg poly(A)). 2. For each different probe used, include two control tubes containing the same amount of labeled probe used for the experimental tubes in step 1, plus Yeast RNA equivalent to the highest amount of sample RNA (see Note 8). 3. Add 1/10th volume of 5 M NH4OAc. 4. Add 2.5 volumes of ethanol, mix thoroughly, and allow RNA to precipitate at −20 °C for at least 15 min or overnight (see Note 9). 5. Centrifuge at maximum speed in a microcentrifuge (≥10,000×g) for 15 min at 4 °C (see Note 10). 6. Remove the supernatants carefully and air-dry the pellets for 5 min. 7. Add 10 μl of Hybridization Buffer III to each pellet, vortex each tube briefly, then microfuge for a few seconds. 8. Heat samples to 90–95 °C for 3–4 min to denature the RNA. 9. Vortex tubes after the incubation and microfuge briefly. 10. Incubate at 42 °C overnight for hybridization. 11. Prepare a master mix dilution of RNase in RNase Digestion III Buffer (150 μl buffer and 1.5 μl RNAse A/T1 mixture per reaction). 12. Briefly centrifuge the sample tubes to remove condensation in the tube. 13. Add 150 μl of the RNase mix to each reaction. 14. Vortex and microfuge tubes briefly (see Note 11). 15. Incubate the tubes for 30 min at 37 °C. 16. Add 225 μl RNase Inactivation Solution III and incubate for 15 min at −20 °C. 17. Centrifuge the tubes for 15 min at maximum speed at 4 °C. 18. Carefully remove all supernatant from each tube and air-dry the pellets for 15 min. The gel size and acrylamide concentration will be dictated by the experiment; specifically, the number and sizes of probes, and their relation to each other. A 5 % acrylamide gel will effectively resolve fragments of about 50–1,000 nucleotides. 1. Prepare 45 ml 5 % acrylamide gel using 21.6 g urea, 4.5 ml 10× TBE, 5.7 ml 40 % acrylamide (acryl–bis-acryl=19:1) and water to 45 ml. 3.3 Separation and Detection of Protected Fragments Single Nucleotide RNA Mapping
- 46. 32 2. Stir at room temperature until the urea is completely dissolved. 3. Follow the manufacturer’s instructions for the details of attaching the glass plates to pour the gel. 4. Add 60 μl 10 % ammonium persulfate to gel solution. 5. Start the polymerization by adding 48 μl TEMED and mix briefly. 6. Start to pour gel immediately and wait 60 min to complete polymerization. 7. Follow the manufacturer’s instructions to set up the gel and S2 apparatus. 8. Use 1× TBE as the gel running buffer. 9. It is very important to rinse the wells of urea-containing gels immediately before loading the samples. 10. Heat the samples at 85 °C for 5 min and chill on ice before loading to gel. 11. Add appropriate RNA size marker or sequence marker to a single well for RNA mapping experiments (see Note 12). 12. Run the gel about 60 W. 13. Dry gel after the run using a vacuum gel dryer. 14. Use autoradiography or digital radioactive imaging system to assess the recovered product. 15. If you detect no or too many fragments like a smear, adjust assay conditions according to Table 1. 4 Notes 1. To synthesize a labeled antisense RNA probe from DNA tem- plates in vitro, SP6, T3, and T7 phage RNA polymerases are widely used. The template must have a double-stranded 19–23 base promoter upstream of the sequence to be transcribed. Many commercially available cloning vectors contain two or more separate phage promoters flanking a multiple cloning site. 2. The MAXIscript Kit can be used to incorporate virtually any labeled nucleotide into RNA. Traditionally 32P labeled UTP or CTP has been used in the MAXIscript Kit, but other isoto- pically labeled nucleotides (33P, 35S, 3H) can be used with this kit as well. 3. The spermidine in the 10× Transcription Buffer can precipitate. 4. 10× Transcription Buffer can coprecipitate the template DNA if the reaction is assembled on ice. 5. Incubate reactions with 3–10 μM limiting nucleotide for 10 min and reactions with >10 μM limiting nucleotide for 1 h. Mahtab Nourbakhsh
- 47. 33 6. DNase digestion is important to remove the template as it can hybridize to the probe and cause false positive signals. 7. By heterogeneous length of probe, it is necessary to isolate primarily full length probe. We recommend purification of probe using polyacrylamide gel as described in manufacturer’s protocol. 8. Yeast RNA is not an appropriate control if the probe is expected to hybridize with sequences found in yeast RNA. 9. Extended storage of radiolabeled probes will result in radiolysis. 10. To make it easier to locate the pellets, it is helpful to position all the tubes with the hinges of the lids facing away from the center of rotation. The pellets will all form directly below the hinges. Table 1 Troubleshooting guide Problem Recommendations The target is not present in the sample Overdigestion with RNase is rarely seen and would lead to smearing of the signal below the expected position of the protected fragment Confirm that absence of signal is a legitimate result. Use a separate sample known to contain detectable levels of the target RNA. If this is not possible, in vitro synthesized sense-strand RNA can be added to a RNA samples to serve as a positive control. Increase the sensitivity of the assay using a longer probe with a higher specific activity, or increase the amount of target RNA up to about 50 μg of total RNA per hybridization reaction in the assay. In some cases better results can be achieved by higher hybridization temperatures Smear or ladder in the no-target/no-RNase control lane Gel wells might be overloaded which reduces the resolution of the bands. Use wider gel wells Degradation of probe is possible. This is most often from radiolysis, but it can also be due to RNase contamination of the probe solution, your tubes, or pipette tips. Resynthesize the probe, avoid contamination and do not store the prober at −20 °C longer than a week Full-length probe is seen in all lanes RNase(s) were completely inactive or were omitted. Use a new batch of enzyme Too much probe might be added to the reaction. No more than 2–8×104 cpm of high specific activity probe should be used for up to 10 μg of RNA Residual DNA is protecting the probe from digestion. Use less DNA template in transcription reaction. Alternatively, transcription reaction can be treated with RNase-free DNase I before hybridization Aberrant, pointed, or smeared bands appear The supernatant from the final precipitation step might be not completely removed. Salt may lead to aberrant migration (“tunneling”) of the protected fragment. We recommend removing the residual supernatant thoroughly using a very fine-tipped pipette Single Nucleotide RNA Mapping
- 48. 34 11. It is important to have no-RNase control tube(s) which serves as a control for probe integrity. It will also show the gel migration of the full-length probe. If there is any unexpected degradation of the probe, it will be seen in this control. Ideally, this lane should show a single band. 12. The basic requirement for RNA mapping is that the probe spans the region to be mapped. This usually means that the probe is derived from a genomic clone, as opposed to a cDNA clone. For example, in order to map the transcription initiation site for a given mRNA, a probe is prepared by subcloning and transcribing a genomic fragment that extends from upstream of the gene of interest to some point in the first exon. Probe synthesis, purification, hybridization, and RNase digestion are carried out using the standard RNase protection assay. The transcription start site is mapped by comparing the size of the protected fragment to the size of the undigested probe. For exact determination of the size of the protected fragment, the sample is analyzed on a gel in conjunction with a sequencing reaction of the RNA probe or RNA maker. References 1. Ma C, Wang J, Li L, Duan MJ, Zhou YH (2011) Identification of true EST alignments for recog- nising transcribed regions. Int J Data Min Bioinform 5:465–484 2. Hobbs MV, Weigle WO, Noonan DJ, Torbett BE, McEvilly RJ, Koch RJ, Cardenas GJ, Ernst DN (1993) Patterns of cytokine gene expression by CD4+ T cells from young and old mice. J Immunol 150:3602–3614 3. Kekule AS, Lauer U, Meyer M, Caselmann WH, Hofschneider PM, Koshy R (1990) The pre S2/S region of integrated hepatitis B virus DNA encodes a transcriptional transactivator. Nature 343:457–461 4. Gelfman S, Ast G (2013) When epigenetics meets alternative splicing: the roles of DNA methylation and GC architecture. Epigenomics 5:351–353 Mahtab Nourbakhsh
- 49. 35 M. Lucrecia Alvarez and Mahtab Nourbakhsh (eds.), RNA Mapping: Methods and Protocols, Methods in Molecular Biology, vol. 1182, DOI 10.1007/978-1-4939-1062-5_4, © Springer Science+Business Media New York 2014 Chapter 4 Analysis of RNA Secondary Structure Mahtab Nourbakhsh Abstract RNA has different levels of structural organization. The primary structure is the linear order of the nucleotide monomers, the RNA sequence. During transcription process, the partially synthesized RNA is folded by base-pairing and thermodynamic intramolecular or intermolecular interactions. This results in a dynamic spreading of a secondary structure along the length of the transcribed section of the RNA. The analysis of both primary or secondary structures requires the RNA end-labeling either at its 5′ end using a kinase reaction with [gamma-32P]ATP, or at its 3′ end using an RNA ligation reaction with [32P]pCp. End-labeled RNAs are then gradually breakdown using hydrolysing chemicals or a variety of enzymes targeting specific RNA sequences and secondary structure. The most commonly used enzymes are RNase A, T1, and V1. The partial digestion of the RNA reveals a mix of truncated RNA fragments of different lengths, called RNA ladder. The products are then separates through a high resolution gel system and subjected to autoradiographic analysis. Each visible fragment is labeled at one end, but comprises an enzyme specific sequence at the other end. Final comparison of the detected RNA ladders reveals a hypo- thetical model of the secondary RNA structure under assay conditions. Key words Secondary structure, Double-stranded RNA, Single-stranded RNA, RNAse, Mfold 1 Introduction RNA molecules possess a variety of single-stranded and double- stranded regions that lead to complex three-dimensional structures. These structures are mostly crucial for the molecule’s interactions with other regulatory molecules like nucleic acids and proteins. Thus, RNA structure plays a central role in many cellular processes, including transcription initiation, elongation and termination, reg- ulation of gene expression, and protein translation. Thus, elucidat- ing the mechanistic aspects of RNA interactions often requires a detailed understanding of the underlying RNA structure. Analysis of RNA structure is traditionally based on successive enzymatic cleavage of folded RNA using specific RNases. First, RNA of interest needs to be synthesized in vitro using available in vitro transcription systems. This step requires a DNA template containing the RNA sequence of interest localized downstream to
- 50. 36 a synthetic promoter, T3, T7, or SP6 (see Note 1). Using the MAXIscript Kit described in this chapter, high amount of RNA can be synthesized in a 10-min reaction using corresponding polymerase. Next, the RNA product needs to be labeled at 5′ or 3′ end using a kinase reaction with [gamma-32P]ATP or an RNA ligation reaction with [32P]pCp, respectively. T4 Polynucleotide Kinase (PNK) catalyzes the transfer of the gamma-phosphate of ATP to the 5′-hydroxyl termini of RNA. This phosphate transfer is commonly referred to as a kinase or phosphorylation reaction. RNAs with a 5′-hydroxyl (OH) group can be added directly to a kinase reaction. However, RNAs with a 5′-phosphate should be dephosphorylated first using Calf Intestinal Phosphatase prior to labeling with PNK and [gamma-32P]ATP. T4 RNA ligase catalyzes the ligation of the 5′ phosphate termi- nus of a nucleic acid donor to the 3′ OH terminus of a nucleic acid acceptor. The reaction is ATP dependent, and the 3′ end of target RNA is labeled by adding [32P]pCp to the reaction. The most commonly used RNases for performing RNA structural analysis are RNases A, V1, and T1. These enzymes bind to specific sequences and cleave folded RNA at specific sequence patterns. RNase A is a pancreatic ribonuclease that cleaves the target RNA 3′ of single-stranded C and U residues. It cleaves the phosphodiester bond between the 5′-ribose of a nucleotide and the phosphate group attached to the 3′-ribose of an adjacent pyrimidine nucleotide. The resulting 2′, 3′-cyclic phosphate is hydrolysed to corresponding 3′-nucleoside phosphate. RNase V1 is a metal-dependent ribonuclease specific for dsRNA regions of 4 nucleotides or more. Cleavage occurs between the 3′-hydroxyl of any ribonucleotide and the 5′-phosphate group of the adjacent ribonucleotide. Commercially available RNase T1 is isolated by a series of purification steps from recombinant E. coli strains overex- pressing the RNase T1 gene of Aspergillus oryzae. The purified enzyme specifically cleaves the target RNA 3′ to single-stranded guanosine residues, producing 3′-phosphorylated ends. To facilitate RNA structural studies, the exposure time of RNA to RNase is strictly limited allowing for a single cleavage per RNA strand. The end products of RNase reactions comprise of labeled RNA ladders which can be analyzed using high resolution gel elec- trophoresis. To help identify the cleavage site locations, another ladder is generated by alkaline hydrolysis which cleaves RNA strand by single nucleotides. In addition to the described experimental analysis here, we recommend the use of available software for predicting RNA structure [1–3]. Secondary structure is the set of the canonical base pairs, and secondary structure can be predicted by compara- tive sequence analysis in silico. The most commonly used method is free energy minimization. The accuracy of structure prediction is then improved either by using experimental mapping data or by Mahtab Nourbakhsh
- 51. 37 predicting a structure conserved in a set of homologous sequences. Additionally, tertiary structure, the three-dimensional arrange- ment of atoms, can be modeled with guidance from comparative analysis and experimental techniques. New approaches are also available for predicting tertiary structure. 2 Materials 1. MAXIscript® kit (Life Technologies). 2. DNA template (see Note 1). 3. Ribonucleotides. 4. Trichloroacetic acid: molecular biology grade. 5. Ethanol: ACS reagent grade. 6. 0.5 M EDTA. 7. Centri-Spin™ 40 Columns (Life Technologies). 8. T4 RNA Ligase (Ambion Cat #2140). 9. 10× T4 RNA Ligase Buffer (supplied with Ambion’s T4 RNA Ligase: 0.5 M Tris–HCl, pH 7.8, 0.1 M MgCl2, 0.1 M DTT, 10 mM ATP). 10. [32P]pCp. 11. RNase-free Sephadex G-25 or G-50 spin columns such as Ambion’s NucAway™ Spin Columns (Cat #10070). 1. Nuclease-free Water. 2. 10× Dephosphorylation buffer (0.5 M Tris–HCl, pH 8.5, 1 mM EDTA, pH 8). 3. Calf Intestinal phosphatase (CIP; 0.1 U/μl). 4. Phosphatase removal reagent (available in the KinaseMax™ Kit). 5. [gamma-32P]ATP (7,000 Ci/mmol, 150 mCi/ml). 6. 10× Kinase buffer (500 mM Tris, pH 7.5, 100 mM MgCl2, 50 mM DTT). 7. T4 Polynucleotide Kinase (10 U/ml). 8. RNase-free Sephadex G-25 or G-50 spin columns such as Ambion’s NucAway™ Spin Columns (Cat #10070). 1. 0.1–3 μg end-labeled RNA. 2. Yeast RNA (10 mg/ml, Ambion Cat # 7118). 3. 1× Alkaline Hydrolysis Buffer (supplied with Ambion’s RNA Grade Ribonucleases: 50 mM Sodium Carbonate [NaHCO3/ Na2CO3] pH 9.2, 1 mM EDTA). 2.1 RNA Synthesis 2.2 RNA 3′ End Labeling 2.3 RNA 5′ Labeling 2.4 RNA Hydrolysis RNA Structure Analysis
- 52. 38 4. 0.2–4 μg end-labeled RNA. 5. Yeast RNA (10 mg/ml, Ambion Cat # 7118). 6. 10× RNA structure buffer (supplied with Ambion’s RNA Grade Nucleases: 100 mM Tris pH 7.0, 1 M KCl, 100 mM MgCl2). 7. RNase T1 (1 U/μl, Ambion Cat # 2283). 8. Inactivation/precipitation buffer (supplied with Ambion’s RNA Grade Nucleases). 9. 100 % ethanol. 1. Acrylamide Gel Loading Buffer (Supplied with Ambion’s RNA Grade Ribonucleases: 95 % Formamide, 18 mM EDTA, 0.025 % SDS, 0.025 % Xylene Cyanol, 0.025 % Bromophenol Blue; or Gel Loading Buffer II Cat #8546G). 2. Urea (high quality). 3. 40 % Acrylamide (acryl–bis-acryl=19:1). 4. 10× TBE (0.9 M Tris base, 0.9 M Boric Acid, 20 mM 0.5 M EDTA). 5. 10 % ammonium persulfate. 6. EMED. 7. Vertical S2 Gel Electrophoresis Apparatus Life Technologies. 8. Power supply. 9. Gel Dryer Model 583 Bio-Rad. 3 Methods 1. Thaw the frozen reagents, mix, and microfuge briefly to pre- vent loss and/or contamination of material by opening the lid. 2. Keep all the reagents on ice except the 10× Transcription Buffer. 3. Vortex the 10× Transcription Buffer several times at room temperature until it is completely in solution (see Note 2). 4. Assemble transcription reaction according to manufacturer’s recommendations at room temperature by adding the DNA, water, nucleotides, and 10× Transcription Buffer (see Note 3). 5. Mix thoroughly by pipetting the mixture up and down gently. 6. Incubate the reaction for 10 min to 1 h at 37 °C. 7. Add 1 μl TURBO DNase, mix well, and incubate at 37 °C for 15 min (see Note 4). 8. Add 1 μl of 0.5 M EDTA to stop the reaction to inactivate DNase and block the heat-induced RNA degradation. 9. Purify the transcripts using Centri-Spin™ 40 Columns (see Note 5). 2.5 RNA Enzymatic Digestion 2.6 High Resolution Gel Analysis 3.1 RNA Synthesis Mahtab Nourbakhsh
- 53. 39 1. Combine 2 μl 10× T4 RNA Ligase Buffer, 50–100 pmol RNA, equimolar amount (50–100 pmol) [32P]pCp and RNase-free water to a final volume of 18 μl in a single RNase- free microfuge tube. 2. Mix thoroughly by pipetting the mixture up and down gently. 3. Add 2 μl T4 RNA Ligase (10 U) and mix thoroughly by pipet- ting the mixture up and down gently. 4. Incubate at 4 °C overnight (10–12 h). 5. Remove unincorporated nucleotides by applying the mixture to an RNase-free Sephadex G-25 or G-50 spin column (e.g., NucAway Spin Columns) following the manufacturer’s recommendations. 6. If the RNA is not labeled efficiently, follow the instructions in Table 1. 1. Combine Nuclease-free Water to make a final volume of 10 μl, 0.1–10 pmol RNA, 1 μl 10× dephosphorylation buffer (0.5 M Tris–HCl, pH 8.5, 1 mM EDTA, pH 8) and 1 μl Calf Intestinal Phosphatase (CIP; 0.1 U/μl) in a single RNase- free microfuge tube. 2. Mix thoroughly by pipetting the mixture up and down gently. 3. Incubate for 1 h at 37 °C. 4. Remove the Calf Intestine Alkaline Phosphatase by use of the Phosphatase Removal Reagent (available in the KinaseMax™ Kit). 5. Combine nuclease-free water to make a final volume of 20 μl, 25 pmol [gamma-32P]ATP (7,000 Ci/mmol, 150 mCi/ml), 2 μl 10× Kinase Buffer (500 mM Tris, pH 7.5, 100 mM MgCl2, 50 mM DTT), and 1 μl T4 Polynucleotide Kinase (10 U/ml). 6. Mix thoroughly by pipetting the mixture up and down gently. 7. Incubate at 37 °C for 1 h. 8. Remove unincorporated nucleotides by applying the mixture to an RNase-free Sephadex G-25 or G-50 spin column (e.g., NucAway Spin Columns) following the manufacturer’s recommendations. 9. If the RNA is not labeled efficiently, follow the instructions in Table 1. This procedure provides a gel electrophoresis “ladder” of hydro- lyzed RNA fragments. In the procedure, three different hydrolysis times are used. After the experiment, select the ladder that pro- vides the best distribution of nucleic acids over the range of lengths needed for your experiments. 1. Mix 0.1–3 μg of end-labeled RNA and 3 μg of yeast tRNA in 5 μl or less. 3.2 RNA 3′ End Labeling 3.3 RNA 5′ End Dephosphorylation and 5′ End Labeling 3.4 RNA Secondary Structure Analysis 3.4.1 Alkaline Hydrolysis RNA Structure Analysis
- 54. 40 2. Add sufficient 1× Alkaline hydrolysis buffer to bring the final volume to 15 μl. 3. Aliquot 5 μl of the RNA–buffer mixture into three tubes labeled 1–3. 4. Heat the tubes to 95 °C to denaturation. 5. After 2 min, remove Tube #1 to an ice bucket. 6. After 5 min, remove Tube #2 to an ice bucket. 7. After 15 min, remove Tube #3 to an ice bucket. 8. Add 10 μl of Acrylamide Gel Loading Buffer to each of the three tubes. For an untreated control, mix 1 μl of 5′ end- labeled RNA with 8 μl of Acrylamide Gel Loading Buffer. Table 1 Troubleshooting guide Problem Recommendations Samples do not label well The quality of the RNA preparation is a crucial factor in the labeling reaction. Make sure your RNA does not contain small RNA or DNA fragments, residual salts, or monovalent cation concentrations ≥100 mM. You may clean up the RNA by spin-column purification Your RNA might form a complex or strong tertiary structure decreasing the efficiency of the CIP or kinase reaction. Preheating the RNA to 90 °C for 2 min and immediate transfer on ice might dissolve any secondary structure in the RNA The abundance of detected fragments is very low The RNAses are optimized and purified for RNA structure analysis, RNA end mapping experiments. The enzymes cannot be contaminated with other nucleases causing unexpected cleavage sites. Thus, we recommend that the concentration of target RNA and enzymes need to be optimized in individual experiments. Reduce the concentration of the nucleases to increase the abundance of cleavage products Identical fragments observed by 5′ labeled RNAs independent of nuclease concentration or enzyme In most cases, this is caused by unexpected termination of transcription during probe synthesis based on truncated templates. Examine the integrity and length of the DNA template In some cases a strong tertiary structure can cause a nick in the RNA which might affect the run pattern of fragments in gel analysis. Examine the denaturation step and temperature. You may denaturate the samples at higher temperature or for an extended time No RNA fragments can be observed in all reactions This indicates the RNAse contamination in the synthesized RNA. In this case you will need to start all over again and prepare new DNA template Mahtab Nourbakhsh
- 55. Another Random Document on Scribd Without Any Related Topics
- 56. The great objects of this Government are contained in the context of the constitution. He recapitulated those objects, and inferred that every power necessary to secure these must necessarily follow; for as to the great objects for which this Government was instituted, it is as full and complete in all its parts as any system that could be devised; a full, uncontrollable power to regulate the fiscal concerns of this Union, is a primary consideration in this Government, and from hence it clearly follows that it must possess the power to make every possible arrangement conducive to that great object. He then adverted to the late Confederation, and pointed out its defects and incompetency; and hence the old Congress called on the States to enact certain laws which they had not power to enact; from hence he inferred, that as the late Confederation could not pass those laws, and to capacitate the Government of the United States, and form a more perfect union, the constitution under which we now act was formed. To suppose that this Government does not possess the powers for which the constitution was adopted, involves the grossest absurdity. The deviation from charters, and the infringement of parchment rights, which had been justified on the principle of necessity by the gentleman from Virginia, (Mr. Madison,) he said had been made on different principles from those now mentioned; the necessity, he contended, did not at the time exist; the old Congress exercised the power, as they thought, by a fair construction of the Confederation. On constructions, he observed, it was to be lamented that they should ever be necessary; but they had been made; he instanced the power of removability, which had been an act of the three branches, and has not been complained of. It was at least as important a one as the present. But the construction now proposed, he contended, was an easy and natural construction. Recurring to the collection law, he observed, that it was by construction that the receipts are ordered to be made in gold and silver.
- 57. With respect to creating a mass of capital, he supposed just and upright national measures would create a will to form this capital. Adverting to the idea that Congress has not the power to establish companies with exclusive privileges, he observed, that by the amendments proposed by New Hampshire, Massachusetts, and New York, it plainly appears that these States considered that Congress does possess the power to establish such companies. The constitution vests Congress with power to dispose of certain property in lands, and to make all useful rules and regulations for that purpose; can its power be less over one species of its own property than over another? With respect to giving preference to one State over another, he observed, that ten years hence the seat of Government is to be on the Potomac, and wherever the Government is finally settled, the place will enjoy superior advantages; but still the Government must go thither, and the places not enjoying those advantages must be satisfied. It is said we must not pass a problematical bill, which is liable to a supervision by the Judges of the Supreme Court; but he conceived there was no force in this, as those judges are invested by the Constitution with a power to pass their judgment on all laws that may be passed. It is said that this law may interfere with the State Governments; but this may or may not be the case; and in all interference of the kind the particular interest of a State must give way to the general interest. With respect to the corporation possessing the power of passing laws, this, he observed, is a power incidental to all corporations; and in the instance of the Western Territory, Congress have exercised the power of instituting corporations or bodies politic, to the greatest possible extent. He defended the right of Congress to purchase and possess property, and quoted a passage in the Constitution to show that they
- 58. possess this right. He then touched on the expediency of banks, and of that proposed in particular. The advantages generally derived from these institutions, he believed, applied peculiarly to this country. He noticed the objection from banks banishing the specie; he said the surplus only would be sent out of the country; but is it given away? No, sir, it is sent off for articles which are wanted, and which will enrich the country. With respect to a run on the Bank, he mentioned the circumstances under which those runs on the British banks, which had been noticed, took place; and showed there was no parallel that would probably ever take place in this country. For several particulars he showed that the objection which arose from the United States not having a good bargain by the system was not well founded. He then mentioned the peculiar advantages which the United States will enjoy over common subscribers. The objection from banks being already established in the several States he obviated by stating the mischiefs which might arise from an ignorance of the situation of those banks; and concluded by some remarks on the inexpediency of the General Government having recourse to institutions of merely a local nature. Mr. Jackson said, that having been the person who brought forward the constitutional objection against the bill, he thought himself bound to notice the answers which had been offered to that objection. Newspaper authorities, said he, have been alluded to, and their silence on the subject considered as indicating the approbation of the people. He would meet the gentlemen on that ground; and, though he did not consider newspapers as an authority to be depended on, yet if opinions through that channel were to be regarded, he would refer the gentlemen to those of this city; the expediency and constitutionality of the bill have been called in question by the newspapers of this city.
- 59. The latitude contended for in constructing the constitution on this occasion he reprobated very fully. If the sweeping clause, as it is called, extends to vesting Congress with such powers, and necessary and proper means are an indispensable implication in the sense advanced by the advocates of the bill, we shall soon be in possession of all possible powers, and the charter under which we sit will be nothing but a name. This bill will essentially interfere with the rights of the separate States, for it is not denied that they possess the power of instituting banks; but the proposed corporation will eclipse the Bank of North America, and contravene the interests of the individuals concerned in it. He then noticed the several arguments drawn from the doctrine of implication; the right to incorporate a National Bank has been adduced from the power to raise armies; but he presumed it would not be contended that this is a bill to provide for the national defence. Nor could such a power, in his opinion, be derived from the right to borrow money. It has been asked what the United States could do with the surplus of their revenue without the convenience of a bank in which to deposit it with advantage? For his part, though he wished to anticipate pleasing occurrences, he did not look forward to the time when the General Government would have this superabundance at its disposal. The right of Congress to purchase and hold lands has been urged to prove that they can transfer this power; but the General Government is expressly restricted in the exercise of this power; the consent of the particular State to the purchase for particular purposes only is requisite; these purposes are designated, such as building light-houses, erecting arsenals, &c. It has been said that banks may exist without a charter; but that this incorporation is necessary in order that it may have a hold on the Government. Mr. J. strongly reprobated this idea. He was astonished to hear such a declaration, and hoped that such ideas would prevent a majority of the House from passing a bill that would thus establish a perpetual monopoly; we have, said he, I believe, a perpetual debt;
- 60. I hope we shall not have a perpetual corporation. What was it drove our forefathers to this country? Was it not the ecclesiastical corporations and perpetual monopolies of England and Scotland? Shall we suffer the same evils to exist in this country instead of taking every possible method to encourage the increase of emigrants to settle among us? For if we establish the precedent now before us, there is no saying where it will stop. The power to regulate trade is said to involve this as a necessary means; but the powers consequent on this express power are specified, such as regulating light-houses, ships, harbors, &c. It has been said that Congress has borrowed money; this shows that there is no necessity of instituting any new bank, those already established having been found sufficient for the purpose. He denied the right of Congress to establish banks at the permanent seat of Government, or on those sandheaps mentioned yesterday; for if they should, they could not force the circulation of their paper one inch beyond the limits of those places. But it is said, if Congress can establish banks in those situations, the question becomes a question of place, and not of principle; from hence it is inferred that the power may be exercised in any other part of the United States. This appeared to him to involve a very dangerous construction of the powers vested in the General Government. Adverting to the powers of Congress in respect to the finances of the Union, he observed that those powers did not warrant the adoption of whatever measures they thought proper. The constitution has restricted the exercise of those fiscal powers; Congress cannot lay a poll tax, nor impose duties on exports; yet these undoubtedly relate to the finances. The power exercised in respect to the Western Territory, he observed, had reference to property already belonging to the United States; it does not refer to property to be purchased, nor does it authorize the purchase of any additional property; besides, the powers are express and definite, and the exercise of them in making
- 61. needful rules and regulations in the government of that Territory does not interfere with the rights of any of the respective States. Mr. J. denied the necessity of the proposed institution; and noticing the observation of Mr. Ames, that it was dangerous on matters of importance not to give an opinion, observed that be could conceive of no danger that would result from postponing that construction of the constitution now contended for to some future Congress, who, when the necessity of a banking institution shall be apparent, will be as competent to the decision as the present House. Alluding to the frequent representations of the flourishing condition of the country, he inferred that this shows the necessity of the proposed institution does not exist at the present time; why, then, should we be anticipating for future generations? State banks he considered preferable to a National Bank, as counterfeits can be detected in the States; but if you establish a National Bank, the checks will be found only in the city of Philadelphia or Conococheague. He passed a eulogium on the Bank of Pennsylvania; the stockholders, said he, are not speculators; they have the solid coin deposited in their vaults. He adverted to the preamble and context of the constitution, and asserted that this context is to be interpreted by the general powers contained in the instrument. Noticing the advantages which it had been said would accrue to the United States from the Bank, he asked, is the United States going to commence stockjobbing? The "general welfare" are the two words that are to involve and justify the assumption of every power. But what is this general welfare? It is the welfare of Philadelphia, New York, and Boston; for as to the States of Georgia and New Hampshire, they may as well be out of the Union for any advantages they will receive from the institution. He reprobated the idea of the United States deriving any emolument from the Bank, and more especially he reprobated the influence which it was designed the Government should enjoy by it. He said the Banks of Venice and Amsterdam were founded on different principles. In the famous Bank of Venice, though the Government
- 62. holds no shares, yet it has at command five millions of ducats; but the United States were to be immediately concerned in theirs, and become stockholders. The Bank of Amsterdam was under the entire direction of the burgomasters, who alone had the power of making by-laws for its regulation. This power, by the bill, was given up by Government, very improperly he thought, and was to be exercised by the stock- jobbers. The French Bank, he added, was first established upon proper principles and flourished, but afterwards became a royal bank; much paper was introduced, which destroyed the establishment, and was near oversetting the Government. The facility of borrowing he deprecated; it will involve the Union in irretrievable debts; the facility of borrowing is but another name for anticipation, which will in its effects deprive the Government of the power to control its revenues; they will be mortgaged to the creditors of the Government. Let us beware of following the example of Great Britain in this respect. He said, undue advantages had been taken in precipitating the measure, and the reasonable proposition respecting the State debts is not admitted. This I consider as partial and unjust. A gentleman from Virginia has well observed that we appear to be divided by a geographical line; not a gentleman scarcely to the eastward of a certain line is opposed to the Bank, and where is the gentleman to the southward that is for it? This ideal line will have a tendency to establish a real difference. He added a few more observations, and concluded by urging a postponement, if any regard was to be had to the tranquillity of the Union. Mr. Boudinot said he meant to confine himself to two or three great points on which the whole argument appeared to him to rest. He considered the objections to the bill as pointed against its constitutionality and its expediency. It was essential, he observed, that every member should be satisfied, as far as possible, of the
- 63. first; for however expedient it might be, if it was clearly unconstitutional, the bill should never receive the sanction of the representatives of the people. He would, in a great measure, refer its expediency, if constitutional, to the experience of every gentleman of the House, as the most satisfactory proof on that head, and he conceived there was no need of much argument in support of its decision. The first question then was, is Congress vested with a power to grant the privileges contained in the bill? This is denied, and ought to be proved. In order to show in what manner this subject had struck his mind, he first laid down these principles: Whatever power is exercised by Congress must be drawn from the constitution; either from the express words or apparent meaning, or from a necessary implication arising from the obvious intent of the framers. That whatever powers (vested heretofore in any individual State) not granted by this instrument, are still in the people of such State, and cannot be exercised by Congress. That whatever implication destroys the principle of the constitution ought to be rejected. That in construing an instrument, the different parts ought to be so expounded as to give meaning to every part which will admit of it. Having stated these preliminaries, Mr. B. proceeded to inquire what were the powers attempted to be exercised by this bill? For, until the powers were known, the question of constitutionality could not be determined. By it Congress was about to exercise the power of incorporating certain individuals, thereby establishing a banking company for successfully conducting the finances of the nation. The next inquiry is, what rights will this company enjoy in this new character, that they do not enjoy independent of it? Every individual citizen had an undoubted right to purchase and hold property, both real and personal, to any amount whatever; to dispose of this property to whom and on what terms he pleased; to lend his money on legal interest to any person willing to take the same; and indeed
- 64. to exercise every power over his property that was contained in the bill. Individual citizens, then, having these powers, might also associate together in company or copartnership, and jointly exercising the same rights, might hold lands in joint tenancy, or as tenants in common, to any amount whatever; might put any sum of money into joint stock; might issue their notes to any amount; might make by-laws or articles of copartnership for their own government; and, finally, might set up a bank to any amount, however great, and no authority in the Government could legally interfere with the exercise of these rights. The great difference between this private association of citizens, in their individual capacities, and the company to be created by this bill, and which is held up in so dangerous a light, is, that the one exposes the company to the necessity of using each individual's name in all their transactions; suits must be brought in all their names; deeds must be taken and given in like manner; each one in his private estate is liable for the default of the rest; the death of a member dissolves the partnership as to him; and for want of a political existence the union may be dissolved by any part of its members, and of course many obvious inconveniences must be suffered merely of an official kind. By the bill these difficulties are to be removed by conveying three qualities to them. 1st. Individuality, or constituting a number of citizens into one legal artificial body, capable by a fictitious name of exercising the rights of an individual. 2d. Irresponsibility in their individual capacity, not being answerable beyond the joint capital. 3d. Durability, or a political existence for a certain time, not to be affected by the natural death of its members. These are the whole of the powers exercised, and the rights conveyed. It is true these are convenient and advantageous to the company, but of trifling importance when considered as a right of power exercised by a National Legislature for the benefit of the Government. Can it be of any importance to the State whether a
- 65. number of its citizens are considered, in legal contemplation, as united in an individual capacity, or separately as so many individuals, especially if the public weal is thereby promoted? By their irresponsibility being known, every person dealing with them gives his tacit consent to the principle, and it becomes part of the contract. And by political duration their powers and abilities are limited, and their rights restricted, so as to prevent any danger that might arise from the exercise of their joint natural right, not only as to the amount of their capital, but as to the by-laws they may make for their government. A private bank could make contracts with the Government, and the Government with them, to all intents and purposes, as great and important as a public bank, would their capital admit of it; though they would not possess such qualities as to justify the confidence of Government, by depending on them in a time of danger and necessity. This might put it in the power of any individuals to injure the community in its essential interests by withdrawing the capital when most needed. To prevent this, and many other inconveniences, it is necessary that a bank for the purposes of Government should be a legally artificial body, possessing the three qualities above mentioned. Mr. B. then took up the constitution, to see if this simple power was not fairly to be drawn by necessary implication from those vested by this instrument in the legislative authority of the United States. It sets out in the preamble with declaring the general purposes for which it was formed: "The insurance of domestic tranquillity, provision for the common defence, and promotion of the general welfare." These are the prominent features of this instrument, and are confirmed and enlarged by the specific grants in the body of it, where the principles on which the Legislature should rest after their proceedings are more fully laid down, and the division of power to be exercised by the general and particular Governments distinctly marked out. By the 8th section, Congress has power "to levy taxes, pay debts, provide for the common defence and general welfare, declare war, raise and support armies, provide for and maintain a
- 66. navy;" and as the means to accomplish these important ends, "to borrow money," and finally, "to make all laws necessary and proper for carrying into execution the foregoing powers." Let us, then, inquire, is the constituting a public bank necessary to these important and essential ends of Government? If so, the right to exercise the power must be in the supreme Legislature. He argued that the power was not contained in express words, but that it was necessarily deduced by the strongest and most decisive implication, because he contended that it was a necessary means to attain a necessary end. Necessary implication had led Congress under the power to lay and collect impost and taxes, to establish officers for the collection, to inflict penalties against those who should defraud the revenue, to oblige vessels to enter at one port and deliver in another; subjected them to various ceremonies in their proceedings, for which the owners were made to pay; and he conceived that it was not so great an exertion of power by implication to incorporate a company for the purpose of a bank. He also deduced the right from the power of paying debts, raising armies, providing for the general welfare and common defence, for which they were to borrow money. All these necessarily include the right of using every proper and necessary means to accomplish these necessary ends. It is certain, he said, that money must be raised from the people. This could not be done in sums sufficient for the exigencies of Government in a country where the precious metals were as scarce as in this. The people in general are poor when compared with European nations; they have a wilderness to subdue and cultivate; taxes must be laid with prudence, and collected with discretion; the anticipation of the revenues, therefore, by borrowing money, becomes absolutely necessary. If so, then as the constitution had not specified the manner of borrowing, or from whom the loan was to be obtained, the supreme Legislature of the Union were at liberty, it was their duty, to fix on the best mode of effecting the purposes of their appointment. For it was a sound principle, that when a general power is granted, and the means are not specified, they are left to the discretion of those in whom the
- 67. trust is reposed, provided they do not adopt means expressly forbidden. The public defence, or general welfare, resting on the annual supplies from uncertain revenues, would expose the very existence of the community. It is the duty of those to whom the people have committed this power to prepare in time of peace for the necessary defence in a time of war. The United States are now happily in a state of peace; but it was impossible for any one to say how long it would continue. By prudent management it might be long preserved; but this prudence consisted in being always found in a state of preparation to defend our country. The constitution contemplates this very duty by authorizing Congress to provide for the common defence by borrowing money. Why borrow money? Are not the annual revenues sufficient? It might be so, if nothing was to be attended to but internal wants; but the common defence and general welfare loudly call for that provision which will produce a constant guard on external enemies and internal insurrections. To this necessary end it becomes Congress to provide that the necessary means may be always at hand, by being able to arm their citizens and provide their support while engaged in the defence of their common country. This can be done only by borrowing money, which is usually of citizens or foreigners; if of the first, it must be from individuals or from private banks: will it be prudent to trust to either? Loans from individuals were attempted during the war, when patriotism produced a will in some lenders, and others were glad to get rid of a depreciating paper currency almost on any terms whatever. But even these loans, arising from this paper medium with which the market was glutted, were altogether insufficient; and by one change of circumstances every hope was precluded of being any way successful in procuring money from that source. The circumstances of individuals, too, in this country are such, when compared with the wants of a nation, as to render the source too vague and uncertain to rely upon; and it would be a most improvident execution of the powers granted for the express purpose of the common defence and general welfare. Private banks are almost as inadequate to the
- 68. object, and for reasons already given, were neither to be depended on for will or capital as to the supply for the principal wants of Government. They are generally established for commercial purposes, and on capitals not always sufficient for them. If they should be prevailed upon at any time to attempt to supply the demands of a nation at war, it must be from a general combination of their whole stocks, to the destruction of the original designs of their several institutions. This ought not to be expected; for as far as it goes to the depression of the mercantile interests, so far it is injurious to the Government; besides, a dependence upon such a combination would be impolitic, both from its slowness and uncertainty. The votes of a few individuals affected by local, selfish, or adverse politics, might endanger the whole people. Such a dependence ought not to be attributed to the wise framers of the constitution, neither does the language warrant it. But foreign loans have been mentioned, as a proper source for this purpose. The imprudence of placing the common defence of a nation on the will of those who have no interest in its welfare is a good answer to this observation. Would it be prudent to trust a foreigner, perhaps a rival, if not an enemy, with your supply of what has emphatically been called the sinews of war? Would it not expose us to exorbitant demands, and often a refusal? Many adventitious circumstances of a war, increasing demands from all quarters, scarcity of coin, and difficulty of communication, as well as the intrigues of courts, all loudly oppose the measure, as contrary to the spirit and meaning of a provision for the common defence and general welfare. The only resort then, he conceived, was by a timely provision to secure institutions at home from which loans might be obtained at all times on moderate terms, and to such amount as the necessity of the State might require. But gentlemen say that the constitution does not expressly warrant the establishment of such a corporation. If by expressly, express words are meant, it is agreed that there are no express words; and this is the case with most of the powers exercised by Congress; for if the doctrine of necessary implication is rejected, he did not see what the supreme Legislature of the Union could do in that character. If this power is not clearly given in the
- 69. constitution by necessary implication, then is a necessary end proposed and directed, while the common and usual necessary means to attain that end are refused, or at least not granted. Mr. B. was firmly of opinion that a National Bank was the necessary means, without which the end could not be obtained. Theory proved it so in his opinion, and the experience of the Union in a day of distress had fully confirmed the theory. The struggles of the friends of freedom during the late contest had nearly been rendered abortive for want of this aid. That danger which was then so hardly avoided became a solemn memento to this House to provide against a similar case of necessity. This was the time to do it with advantage, being in such profound peace. He had not heard any argument by which it was proved that individuals, private banks, or foreigners, could with safety and propriety be depended on as the efficient and necessary means for so important a purpose. Although money was at present plentiful in Europe, and might be borrowed on easy terms, it might not be so to-morrow, in case a war should break out, and our necessities become pressing. He again enumerated the harmless qualities with which it was proposed to vest the bank corporation, by the bill on the table, for the important purposes of the common defence and general welfare. Gentlemen had not yet pointed out any danger arising to the community, neither did he think it possible that any could ever be mentioned equal to those of suffering the Government to depend on individuals or private banks for loans in a day of distress. But it was said that this bill gave the corporation a right to hold real property in a State, which Congress had no power to do. The terms of the bill are misapprehended; this is a right which has been already shown, attaches to the citizens individually, or in their associated capacity; the bill, therefore, does no more than to vest a number with an artificial single capacity under a fictitious name, and by that name to hold lands, make by-laws, &c.; all which they might have done before as citizens in a collective capacity. So far from giving a new power, their original individual rights are limited for the
- 70. public safety as to the amount of their stock and the duration of their existence. Mr. B. then proceeded to cite numerous instances of powers exercised by Congress during the last two years, deduced under the constitution by necessary implication, to show the utter impossibility of carrying any one provision of that authority into execution for the benefit of the people without this reasonable latitude of construction. He also adverted to some instances of the like conduct under the former Confederation. It had been urged that the new Congress had no rights or powers but what had been vested in and given to them by the individual States, and therefore they could not accept a cession from Great Britain by the treaty of peace of the lands extending to the Lake of the Woods, because not before included in any individual State. Every member was soon convinced of the absurdity of the argument, and by a necessary implication established the power of the Confederated Legislature. During the war the Commander-in-chief gave a passport to a British officer to transmit clothing to the British prisoners at Lancaster. He accordingly conveyed a very large quantity of British goods into Pennsylvania for that purpose; which being directly against an express law of that State, they were seized and condemned by the proper magistrate. On a complaint to the Legislature of the State, they referred the same to their Judicial officers, upon whose report (that Congress being vested with the power of declaring war, the right of giving safe passports to an enemy was necessarily implied, which, therefore, was duly exercised by their Commander-in-chief, though no express power was given to him for that purpose) the Legislature declared their law directing the condemnation of the goods void ab initio, and the judgment of condemnation had no effect. This was also the rule that governed this House with regard to the removability of officers by the President, and the authority given to a Council to legislate for the Western Territory. In fine, he concluded, that it was universally understood that whenever a general power was given, especially to a supreme Legislature, every necessary means to carry it into execution were necessarily included. This was
- 71. the common sense of mankind, without which it would require a multitude of volumes to contain the original powers of an increasing Government that must necessarily be changing its relative situation every year or two. If power was given to raise an army, the making provision for all the necessary supplies and incidental charges was included. If a navy was to be formed, the manning and supplying the warlike stores are necessarily included. If a power is given to borrow money, a right to mortgage or pledge the public property to secure the repayment is understood to be vested in the borrower. Take up the present statute book, and every page will afford evidence of this doctrine. Examine the law with regard to crimes and punishments; under the power of establishing courts, we have implied the power of punishing the stealing and falsifying the records, and ascertained the punishment of perjury, bribery, and extortion. Under the power of regulating trade, we have accepted cessions of real estate, and built light- houses, piers, &c. All this is under the doctrine of necessary implication for the public good; and in cases not so strong as the present, and on the exercise of which no gentleman thought proper to start this objection. This construction appears so natural and necessary, that the good sense of every gentleman on the floor has hitherto led him to proceed on this principle ever since we began to legislate; what principle of the constitution does it destroy? It gives nothing that can affect the rights of any State or citizen. Indeed, it has been said that it is exercising a high act of power; he thought it had been shown to be rather of the inferior kind; but allow the position, and who so proper as the Legislature of the whole Union to exercise such a power for the general welfare? It has also been said that this power is a mere conveniency for the purpose of fiscal transactions, but not necessary to attain the ends proposed in the constitution. This is denied, and at best is mere matter of opinion, and must be left to the discretion of the Legislature to determine.
- 72. Mr. B. said, he should now conclude what he had to say, had not an honorable gentleman (Mr. Jackson) brought forward the observations of the author of the Federalist, vol. 2, p. 72, 73, 74, to show a different contemporaneous exposition of the constitution, and charged the author, who he alleged was said to be also the author of the present plan before the House, with a change of sentiment. As this gentleman is not here to speak for himself, he ought to have the next best chance by having what he then wrote candidly attended to, especially as gentlemen allow him to be a good authority. Mr. B. read only part of the 73d page referred to by Mr. Jackson, in these words: "Had the Convention attempted a positive enumeration of the powers necessary and proper for carrying their other powers into effect, the attempt would have involved a complete digest of laws on every subject to which the constitution relates; accommodated, too, not only to the existing state of things, but to all the possible changes which futurity may produce; for in every new application of a general power, the particular powers which are the means of attaining the general power must always necessarily vary with that object, and be often properly varied whilst the object remains the same." How these sentiments can be said to be a different contemporaneous exposition must be left to the House to determine. Mr. B. then begged the indulgence of the House to hear the same gentlemen when arguing expressly on that part of the constitution now under consideration; and then read pp. 144, 145, and 146, of the 1st vol. of the Federalist, which are too long to be inserted. He declared that, in his opinion, it was impracticable to put together language in the same length that could more forcibly and pointedly elucidate and prove the construction contended for in support of the bill on the table. There remained yet but two objections, to answer which Mr. B. would detain the House a little longer. The gentleman from Georgia (Mr. Jackson) had charged the measure with establishing the commercial interests, to the great injury of the agricultural. If this was true he never would agree to it, for he considered the agricultural interests of America as its great and sure dependence. Mr. B. confessed that so far from seeing these
- 73. measures in this point of light, he could not bring his mind to comprehend how the commercial interests of a country could be promoted without greatly advancing the interests of agriculture. Will the farmer have any temptation to labor, if the surplus of what he raises beyond his domestic consumption is to perish in his barn for want of a market? Can a market be obtained without the merchant? If commerce flourishes, the merchants increase, and of course the demand for the produce of the land; but if the mercantile interests fail, there is none to export the surplus produced by agriculture. If the farmer should undertake to export his own produce, he could not give his whole attention to his affairs; or, if the merchant should attempt to raise the grain he wanted, he could not carry on his merchandise. The one interest depends on the other; a separation destroys both. But the incapacity of the Bank to extend its influence to the extremes of the Union has been argued from the gentleman never having seen a note of the present Bank of North America in Georgia; he therefore concludes that bank has never been of any service to her agricultural interests. Mr. B. said that he drew very different conclusions from this fact. He supposed that by means of the bank the traders with Georgia had been enabled to send her the precious metals, while the bank paper had answered their purposes nearer home, where it circulated with undoubted credit. He instanced a case of a Philadelphia merchant, who was possessed of £100 in gold, and £100 credit at the bank; the merchant wanted £100 worth of rice of a Georgia planter, and the like value in flour of a Pennsylvania farmer. When he purchased the one of the Georgian, he could safely pay him the whole in gold, while he found the Pennsylvanian would as readily receive the bank paper for his flour; but had there been no bank, he could have purchased but £50 worth of each, and the Georgia and Pennsylvanian both would have gone without a market for the residue. In short, the whole Union may be likened to the body and limbs; you cannot aid or comfort one but the other must be likewise benefited.
- 74. He said it was, however, difficult and impracticable to show that every measure adopted by the Government should have an effect perfectly equal over so extensive a country as that of the United States; it was sufficient if, upon the whole, the measures of Government, taken all together, produced the desired equality. The last objection was, that by adopting this bill we exposed the measure to be considered and defeated by the Judiciary of the United States, who might adjudge it to be contrary to the constitution, and therefore void; and not lend their aid to carry it into execution. This, he alleged, gave him no uneasiness. He was so far from controverting this right in the Judiciary, that it was his boast and his confidence. It led him to greater decision on all subjects of a constitutional nature, when he reflected that if, from inattention, want of precision, or any other defect, he should do wrong, that there was a power in the Government which could constitutionally prevent the operation of such a wrong measure from affecting his constituents. He was legislating for a nation, and for thousands unborn; and it was the glory of the constitution that there was a remedy even for the failures of the supreme Legislature itself. Upon the whole, then, he said, that on taking the power in question in every point of view, and giving the constitution the fullest consideration, under the advantage of having the objections placed in the strongest point of light by the great abilities of the gentlemen in the opposition, he was clearly in favor of the bill; as to its expediency, there could be little doubt in the minds of any gentleman; and unless more conclusive arguments could be adduced to show its unconstitutionality, he should in the end vote for passing the bill. Saturday, February 5. Bank of the United States.
- 75. The House resumed the consideration of the bill for incorporating the Bank of the United States. The question being on the passage of the bill, Mr. Smith observed, that he considered it his duty to offer the reasons which should influence him in giving his vote on this occasion. He had wished amendments to the bill, as some parts of it, he confessed, did not perfectly please him; but his wishes having been overruled, the question now is, whether the bill shall pass? Though he came southward of the Potomac, the principle of the bill met his approbation. It would be a deplorable thing if this Government should enact a law subversive of the constitution, or that so enlightened a body as the Senate of the United States should, by so great a majority as were in favor of this bill, pass a law so hostile to the liberties of this country, as the opposition to this measure have suggested the bank system to be; and it would be very extraordinary if an officer of this Government who has produced a performance explanatory of the constitution, of such celebrity as to be resorted to as an authority, should be so inconsistent with himself as to propose a law entirely subversive of the principles laid down in his able defence of the constitution. He then adverted to the objection drawn from that article of the constitution, that no preference shall be given to one port over another. He showed that the clause was inserted for a particular purpose, and could not be cited as a rule not to be deviated from, as a preference was and must necessarily be given to one port over another. He produced numerous instances in point. In consequence of various clauses in the revenue laws, general regulations sometimes operate partially, and commercial arrangements, apparently unequal, produce the good of the community at large. In reference to construing the constitution, he observed, that the present moment, when the powers of the Government were assailed from various quarters, he conceived the most improper to contract these powers.
- 76. The right to construe the constitution he argued from the principles advanced by Mr. Madison, in the debate on the power of removability, and read sundry observations from Lloyd's Register, made by that gentleman, corroborative of this sentiment. Those arguments, he conceived, applied very aptly to the present subject. Matters of a fiscal nature necessarily devolve on the General Government, and he urged that every power resulting from the acknowledged right of Congress to control the finances of this country must be as necessarily implied as in the case of the power of removability. He then alluded to the expediency of a National Bank. The Secretary gave notice, in his first report, that this plan was in contemplation. Nothing was ever read with greater avidity; and though it is now more than a year since this intimation was given, yet no objections have been offered against it either by the States or by individuals— even the State of North Carolina has not mentioned it. [Here Mr. Bloodworth (if the reporter did not misunderstand) informed Mr. Smith that the report had not been seen by the Legislature of North Carolina.] Mr. Smith said he was sorry for it—and then proceeded to notice some partial quotations, made by Mr. Jackson, from Dr. Smith's Wealth of Nations, against bank systems. He said, he could have wished the gentleman had been more copious in his quotations from that author; if he had, he would have found that that author has fully demonstrated their utility. He noticed the divisions of opinions on the subject of a National Bank in the city of Philadelphia. He supposed ideas of personal advantages induced these opposing sentiments. He, however, thought this subject should be taken up altogether on general principles; and even if its immediate influence should not extend to the extremes of the Union, if the establishment promises a general preponderating advantage, local considerations must be considered in a secondary point of view. The principal inquiry is, will the institution facilitate the management of the finances? This, he thought, had been made apparent. This is the opinion of the
- 77. Secretary of the Treasury, after due and mature consideration of the subject; and he certainly enjoys the best means of forming an opinion; he is at the head of the Fiscal Department, and deservedly enjoys the public confidence. Very little has been offered to disprove his sentiments on this part of the question, and the inexpediency of the measure should be clearly proved before the plan is rejected; for an officer who deservedly enjoys the public confidence is entitled to the support of the Legislature in those plans which are expedient and constitutional. Mr. S. mentioned instances in which Congress exercised power by implication, and observed, that this was necessary to the execution of the duties which devolve on the Government by the constitution. The power to establish a National Bank must reside in Congress, for no individual State can exercise any such power. The right of no particular State is therefore infringed by the institution. It had repeatedly been said, that Philadelphia would derive peculiar advantages from the Bank of the United States, but, he said, if the present plan should fail, it was a question whether the stockholders of the Bank of North America would not derive greater advantages from the necessity which, in that case, Government would be under of resorting to them for loans. The institution, as before observed, is founded on general principles, and will undoubtedly, in its operations, prove of general utility. Mr. Stone said, if, upon questions like the present, he had given pain to members he regarded, they might be assured the pain was reciprocal. Let us cherish mutual toleration. We might conceive that each pursued the system which he advocated from the purest motives. We differ in our ideas of Government, and our sense of the sacredness of the written compact. We varied widely in our opinions of the direction of this Government. The great lesson of experiment would show who is right; but we are influenced in our habits of thinking by our local situations, and, perhaps, the distinct interests of the States we represent. He observed, that upon the present occasion, the opinions respecting the constitution seem to be divided by a geographical line, dividing the continent. Hence it might be
- 78. inferred, that other considerations mixed with the question; and it had been insinuated that it was warped by the future seat of Government. But other causes may be assigned for the diversity of sentiment—the people to the eastward began earliest in favor of liberty. They pursued freedom into anarchy—starting at the precipice of confusion, they are now vibrating far the other way. He said, that all our taxes are paid by the consumers of manufactures; those taxes are all bounties upon home manufactures. The people to the eastward are the manufacturers of this country; it was no wonder that they should endeavor to strengthen the hands of a Government by which they are so peculiarly benefited. It is a fact that the greatest part of the Continental debt has travelled eastward of the Potomac. This law is to raise the value of the Continental paper. Here, then, is the strong impulse of immediate interest in favor of the Bank. He took notice of the distinction made by the plan of the bill, between Continental and State paper. The State paper, on account of partial payments of interest, still remained in the respective States. But this could not, by the present system, be subscribed; so that the Southern States were deprived of the advantage that might have been given to the only paper they have. But if gentlemen charge us with defending the seat of Government, let them remember that this betrays consciousness of an attack. If they believe that this scheme tends to break the faith of the Union pledged to the Potomac, it is no wonder they suppose we oppose it upon that ground. He would not have mentioned this subject, had it not been hinted at. But let the whole of it come forth; let gentlemen consult their own bosoms; let the public decide the truth of his observations. He hoped he should not be suspected of any bias. That so uniform had been his conduct upon all questions, turning upon principles similar to the present, that every member in the House, he believed, had conjectured rightly of the side he would take, before he had uttered a word upon the subject, When implication first raised its head in this House, he started from it as a serpent which was to sting and poison the constitution. He felt in unison with his country. The fears, the opinions, the jealousies of
- 79. individuals and of States, had been explained by a gentleman from Virginia, (Mr. Madison.) He should only remark, that all those who opposed the Government dreaded this doctrine; those who advocated it, declared that it could not be resorted to; and all combined in opinion that it ought not to be tolerated. Never did any country more completely unite in any sentiment than America in this, "that Congress ought not to exercise, by implication, powers not granted by the constitution." And is it not strange? For the admission of this doctrine destroys the principle of our Government at a blow; it at once breaks down every barrier which the Federal constitution had raised against unlimited legislation. He said, that necessity was the most plausible pretext for breaking the spirit of the social compact, but the people of this country have anticipated that pretext. They have said to the Ministers of this country, "we have given you what we think competent powers, but if experience proves them inadequate, we will enlarge them; but, in the mean time, dare not usurp those which we have reserved." It is agreed on all hands, that the power to incorporate the subscribers to a banking company, is not expressly granted, and although gentlemen have agreed that it is implied—that it is an incident, that it is a means for effectuating powers expressly granted, yet they are not agreed as to the particular power to which this is an incident. They admit, that the sweeping clause in the constitution confers no additional power. But if he understood the gentlemen, several of them were of opinion that all governments, instituted for certain ends, draw to them the means of execution as of common right. This doctrine would make ours but a short constitution. [Here he read the preamble and then said:] Here is your constitution! Here is your bill of rights! Do these gentlemen require any thing more respecting the powers of Congress, than a description of the ends of government? And if, of right, they can carry these into effect, will they regard the means, though they be expressly pointed out? But I would ask if there is any power under heaven which could not be exercised within the extensive limits of this preamble?
- 80. The Convention might have stopped here; and there was no need, according to the doctrine of the gentleman, to point out any of the means for the ends mentioned in the preamble. That portion of the constitution which by all America has been thought so important, according to their logic, would become a dead letter; but the preamble, in fair construction, is a solemn compact, that the powers granted shall be made use of to the ends thereby specified. He then reprobated, in pointed terms, the latitude of the principles premised. He said the end of all government is the public good; and if the means were left to legislation, all written compacts were nugatory. He observed, that the sober discretion of the Legislature, which, in the opinions of gentlemen, ought to be paramount, was the very thing intended to be curbed and restrained by our constitution. He then declared, that our form of government not only pointed out the ends of government, but specified the means of execution. He said, we may make war—this would draw to it the power of raising an army and navy, laying taxes, establishing a judiciary, &c. But the spirit of the constitution, in this respect, had been well explained by Mr. Madison, and he should not recapitulate. He said, a gentleman from South Carolina (Mr. Smith) had remarked that all our laws proceeded upon the principle of expediency—that we were the judges of that expediency—as soon as we gave it as our opinion that a thing was expedient, it became constitutional. What then remains of your constitution, except its mode of organization? We may look into it to refresh our memories respecting the times, places, and manner of composing the Government; that, as to the powers of Congress, were he of that gentleman's opinion, he would never look into it again. Gentlemen see the difficulties of their theories, and are obliged to confess that these incidental powers are not easily defined. They rest in the sober discretion of the Legislature. One gentleman (Mr. Ames) has said, no implication ought to be made against the law of nature, against rights acquired, or against power
- 81. pre-occupied by the States; that it is easier to restrain than to give competent powers of execution. Now these notions are hostile to the main principle of our Government, which is only a grant of particular portions of power, implying a negative to all others. It has been shown that the ends of government will include every thing. If gentlemen are allowed to range in their sober discretion for the means, it is plain that they have no limits. By the cabalistic word incident, your constitution is turned upside down, and instead of being a grant of particular powers, guarded by an implied negative to all others, it is made to imply all powers. But, strange to tell, America forgot to guard it by express negative provisions. Is there any difference in effect between lodging general powers in a government, and permitting the exercise of them by subtle constructions? He said there was a difference. In the one case the people fairly gave up their liberty, and stood prepared; in the other, they were unexpectedly tricked out of their constitution. The preceding remarks showed how dangerous is the doctrine of implication, and upon what small data ingenuity can raise the most dangerous superstructure. He should now take a view of these precedents, in the former and present Congress, which are relied on to justify the present measure. 1st. The Bank of North America. Here he stated the distressful and critical situation of America at the period of its establishment; he remarked, that it was at the time of the declension of the Continental money. He showed that there were no powers in the Confederation to which (even according to the reasoning of the other side) this power could be incidental, but what required the vote of nine States; that the ordinance passed by a vote of seven States, which showed that necessity alone gave birth to that measure. He showed the dissimilarity of the situations of the former and this Congress, and the difference in their powers, and, consequently, in the dangers to be apprehended from the encroachment of either.
- 82. 2d. The redemption of our prisoners at Algiers. This comes within the power to regulate trade. If, said he, we are not capable of redeeming, by the best means in our power, our citizens, our trade may be entirely ruined; and hence, the law which would be made for their redemption would be necessary and proper. But, by the constitution, the Executive may make treaties; these may be general, or for a particular object, and the Legislature may effectuate them by grants of money. 3d. We have bought certificates, and not destroyed them. This, they say, is implied from the power of paying the debts. He asked if, before the purchase, the certificates were debts due from the United States? And demanded, if, by the purchase, they were divested of that quality? In my judgment, when a debt is fairly cancelled, it is as much like a payment as need be. 4th. We had no right, except by implication, to give a salary to the Vice President. He had voted against the salary, and had been for a per diem allowance, because he thought the Vice President was viewed by the constitution only as the President of the Senate. But this example fails most palpably, as Congress, in the compensations, are not confined by the constitution either to a particular sum or mode of payment. 5th. Congress have made corporations, and exercised complete legislation in the Western Territory. He said, to answer this case, nothing more was necessary than to read the clause in the constitution which gives to Congress expressly the power to make all the rules and regulations for them. It seemed to him as if gentlemen were inverting the order of things, by making powers where there were none, and attempting to prove express grants to the implications. 6th. Our regulations respecting freighters and owners, and between captains and seamen. He had not those regulations correctly in his memory, but he believed them proper and necessary regulations of commerce.
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