Biofluid miRNA profiling: from sample to biomarker: miRNA and its Role in Human Disease Webinar Series Part 1

Sample to Insight
Biofluid miRNA profiling: from sample to biomarker
Jonathan Shaffer, Ph.D., Senior Scientist, Product Development
Jonathan.Shaffer@qiagen.com

Sample to Insight
Welcome to our four-part webinar series on miRNAs
2
 Part 1: Biofluid miRNA profiling: from sample to biomarker
 Part 2: Meeting the challenges of miRNA research
 Part 3: Advanced miRNA expression analysis
 Part 4: Functional analysis of miRNA
miRNA and its role in human disease
Biomarker Discovery in Biofluids: From Sample to Biomarker

Sample to Insight
Legal disclaimer
Biomarker Discovery in Biofluids: From Sample to Biomarker 3
QIAGEN products shown here are intended for molecular
biology applications. These products are not intended for
the diagnosis, prevention or treatment of a disease.
For up-to-date licensing information and product-specific
disclaimers, see the respective QIAGEN kit handbook or
user manual. QIAGEN kit handbooks and user manuals
are available at www.QIAGEN.com or can be requested
from QIAGEN Technical Services or your local distributor.

Sample to Insight
QIAGEN tools for biomarker discovery4
Agenda
4
Background1
What is your biomarker I.Q.?2
Sample miRNA biomarker project3

Sample to Insight
What is a biomarker?
A characteristic that is objectively
measured and evaluated as an
indicator of normal biologic
processes, pathogenic processes or
pharmacologic responses to a
therapeutic intervention
Characteristic Methodology
Presence of antibodies ELISA
Abnormal bp, blood cell counts, electrolyte blood counts, pressure
Distinct histological indicators microscopy
Abnormal liver function markers biofluid assay
Presence of muscle injury protein markers biofluid assay
Elevated kidney marker: serum creatinine biofluid assay
Gene status or gene expression status qPCR, NGS, array, etc.

Sample to Insight
Cancer biomarker
Personalized medicine
Is this the
optimal drug
for my
cancer?
Predictive
Is it likely to
develop this
cancer?
Prognostic
What type of
cancer is it?
Diagnostic
What’s the
optimal dose
for my
body?
Pharmacodynamics
Will the
cancer
return?
Recurrence

Sample to Insight
Noninvasive biomarker
In order to use a biomarker for diagnostics, the sample must be as easy to obtain as possible
Urine or saliva sample
A drop of blood like those diabetes patients extract
Blood sample taken by a doctor
CSF
Surgical biopsy
Evaluation

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Canonical pathway of miRNA biogenesis
 Transcribed by RNA polymerase II as a long
primary transcript (pri-miRNAs), which may contain
more than one miRNA
 In the nucleus, pri-miRNAs are processed to
hairpin-like pre-miRNAs by the RNase III Drosha
 Pre-miRNAs are then exported to the cytosol by
exportin 5
 In the cytosol, the RNAse III Dicer processes these
precursors to mature miRNAs
 These miRNAs are incorporated in RISC
 miRNAs with high homology to the target mRNA
lead to mRNA cleavage
 miRNAs with imperfect base pairing to the target
mRNA lead to translational repression and / or
mRNA degradation

Sample to Insight
Why miRNA biomarkers?
 Changes in miRNA can be correlated with gene expression changes in
development, differentiation, signal transduction, infection, aging and disease
 miRNA expression analysis is the foundation for these discoveries
miRBase Entries

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Circulating miRNA biomarkers in the press
‫‏‬No shortage of candidate genes
Cho (2011) Front. Gene. 2
MicroRNA Deregulation in cancer Theragnostic and prognostic value Reference
let-7a Decrease in gastric cancer Discriminate gastric cancer from healthy controls Tsujiura et al. (2010)
let-7f Decrease in NSCLC Associated with overall survival in NSCLC Silva et al. (2011)
miR-1 Decrease in NSCLC Associated with overall survival in NSCLC Hu et al. (2010)
miR-10b Increase in breast cancer Associated with metastases in breast cancer Roth et al. (2010)
miR-17 Increase in gastric cancer Discriminate gastric cancer from healthy controls Zhou et al. (2010)
miR-17 + 106a Increase in gastric cancer Discriminate gastric cancer from healthy controls Zhou et al. (2010)
miR-17-3p Increase in CRC Discriminate CRC from healthy controls Ng et al. (2009)
miR-17-5p Increase in gastric cancer Discriminate gastric cancer from healthy controls Tsujiura et al. (2010)
miR-20b Decrease in NSCLC
Associated with advanced stages and lymph node
metastases in NSCLC
Silva et al. (2011)
miR-21
Increase in CLL harboring 17p deletion
Increase in gastric cancer
Associated with overall survival in CLL
Discriminate gastric cancer from healthy controls
Rossi et al. (2010)
Tsujiura et al. (2010)
miRs-21 + 126 + 210 + 486-5p Deregulate in NSCLC Discriminate stage I NSCLC from healthy controls Shen et al. (2011)
miRs-21 + 155 + 196a + 210 Increase in pancreatic adenocarcinoma
Discriminate pancreatic adenocarcinoma from
healthy controls
Wang et al. (2009)

Sample to Insight
Analytes:
Cell-free miRNA,
mRNA, DNA
Samples:
Serum, plasma,
cerebrospinal fluid (CSF)

Sample to Insight
What is blood?
‫‏‬RBC, WBC, platelets, CTC, “other‫‏‬cells”,‫‏‬extracellular?
RBC, WBC,
platelets, other cells
(e.g., circulating tumor cells)
Serum (post clotting)
Plasma (no-clotting)
 High levels of nucleases present in plasma
 Freely circulating nucleic acids should be rapidly degraded
 Surprisingly, stable nucleic acids can be detected in serum and plasma

Sample to Insight
Stable miRNAs in circulation
‫‏‬An evolving story
1) Valadi, H., et.al.,(2007) Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells, Nat Cell Biol
9:654-659
2) Hunter MP et. al., (2008) Detection of microRNA Expression in Human Peripheral Blood Microvesicles, PLoS ONE 3:e3694
3) Kosaka, N et. al (2010) Secretory mechanisms and intercellular transfer of microRNAs in living cells, J Biol Chem 285: 17442-17452
4) Arroyo, JD et. al., (2011) Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma, Proc. Natl. Acad.
Sci 108: 5003-5008
5) Vickers, KC., et. al., (2011) MicroRNAs are transported in plasma and delivered to recipient cells by high-density lipoproteins. Nat Cell Biol 13:423
6) Wang K, Zhang S, Weber J, Baxter D, Galas DJ.(2010) Export of microRNAs and microRNA-protective protein by mammalian cells. Nucleic Acids Res.
2010 Nov 1;38(20):7248-59.

Sample to Insight
Exosomes / microvesicles (MVs)
It’s‫‏‬the‫‏‬cargo‫‏‬that‫‏‬matters
Blood Plasma
 MVs/exosomes are ~50–200 nm
small vesicles excreted by all
cells
 MVs/exosomes are found in all
biofluids (e.g., blood)
 MVs/exosomes contain stable
RNA (mRNA, miRNA, other
small RNAs), DNA and protein
 Contents are specifically
packaged
 Mechanism of local and distant
cellular communication
 Promise for disease detection
and monitoring
 Use exosomal miRNA profiling in
the absence of tissue to
accurately reflect‫‏‬the‫‏‬tumor’s‫‏‬
profile

Sample to Insight
Should you only look at exosomes?
‫‏‬No‫‏…‏‬well,‫‏‬sometimes!
 Potentially 90% of miRNAs in circulation are present in a non-membrane-bound
form consistent with a ribonucleoprotein complex
 At the same time, analyzing the exosome fraction could maximize signal-to-noise
ratio of a potential biomarker, providing better sensitivity

Sample to Insight
Agenda
16
Background1
Isolationa
Quantificationb

Sample to Insight
PAXgene® Blood miRNA System
‫‏‬Collect / stabilize and purify total RNA from whole blood
Collection/Stabilization: PAXgene Blood RNA Tubes
 Draw volume: 2.5 ml
 Preservation of cellular RNA at point of collection
 Sample stabilization during transport and storage
 Up to 3 days at 18–25°C
 Up to 5 days at 2–8°C
 Seven years storage at –20°C or –80°C (studies ongoing)
Purification: PAXgene Blood miRNA Kit
 High purity RNA from PAXgene-stabilized whole blood
 High yields of small RNA species
 Manual and automated solutions

Sample to Insight
Total RNA (miRNA + mRNA) isolation from cell-free body fluids
‫‏‬miRNeasy Serum / Plasma Kit
 Includes synthetic RNA control assay for normalization
 Minimal elution volume (14 µl)
 High-purity RNA suitable for all downstream applications
 Easy, robust procedures
 Automatable protocol
 When possible, avoid heparanized plasma
 If you can‘t avoid heparinized samples, let me know! We
have a solution!
Purification of circulating RNA from
plasma, serum, CSF, saliva, urine, etc.

Sample to Insight
Exosome purification and total RNA isolation from serum / plasma
‫‏‬exoRNeasy Serum/Plasma Maxi Kit
‫‏‬exoRNeasy Serum/Plasma Midi Kit
 Specifically enriches for RNA contained in vesicles
 Quickly isolates purified total RNA from microvesicles
 Efficiently isolates mRNA and miRNA from plasma/serum
 Enables use of high input volumes for sensitive detection of
low abundance transcripts
 Maxi: 4 ml
 Midi: 1 ml

Sample to Insight
Intact vesicles are eluted from the exoEasy column
Scanning EM (20000x magnification) reveals higher purity with exoEasy
 Both preparations contain vesicle-shaped structures within an expected size range
 UC: Many smaller, unidentified structures/particles that do not match the expected size
 exoEasy: Intact vesicles with higher purity
Ultracentrifugation (UC) Eluate from exoEasy

Sample to Insight
exoRNeasy Serum / Plasma Maxi Kit
‫‏‬Workflow
 Microvesicle
isolation
 20 minutes
 RNA isolation
 35 minutes
Separate serum /
plasma
Isolate
exosomes
Isolate
RNA

Sample to Insight
Agenda
22
Background1
Isolationa
Quantificationb

Sample to Insight
Three phase workflow: sample to classifier
Phase I: Qualitatively determine expressed miRNAs
Pooled samples
miRNome profiling (or NGS)
Phase II: Quantitatively determine differentially expressed miRNAs
Individual profiling of samples (that went into pools)
Screen only expressed miRNAs from Phase I
Phase III: Develop and test classifier model
Addition individual profiling of samples
Screen differentially expressed miRNAs from Phase II

Sample to Insight
miScript PCR System
Complete miRNA quantification system
1. miScript II RT Kit
 HiFlex Buffer: Unparalleled flexibility for miRNA and
mRNA quantification from a single cDNA preparation
 HiSpec Buffer: Unmatched specificity for mature
miRNA profiling
2. miScript miRNA PCR Arrays
 miRNome
 Pathway-focused
3. miScript PreAMP Kit
 Optional step for small or precious samples
 Full miRNome profiling from as little as 1 ng RNA
4. Assays
 miScript Primer Assays
 miScript Precursor Assays
 QuantiTect Primer Assays
5. miScript SYBR Green PCR Kit
 QuantiTect SYBR Green PCR Master Mix
 Universal Primer
6. miScript miRNA PCR Array data analysis software
 Straightforward, free data analysis

Sample to Insight
miScript PCR System
‫‏‬Workflow
1. Isolate total RNA
2. Perform reverse transcription
3. Prepare PCR pre-mix
4. Load PCR arrays or plates
5. Perform real-time PCR
6. Analyze data

Sample to Insight
miRNA expression profiling: miScript miRNA PCR Arrays
‫‏‬Wet-lab-verified miScript Primer Assays pre-dried in PCR plates
 miRBase Profiler miRNome Arrays
 Most species
 Largest content
 High content (HC) arrays
 Targeted miRNome profiling
 Focused arrays
 Bioinformatic-driven profiling
 Formats
 96-well, 384-well, Fluidigm® BioMarkTM
 Compatible with virtually all mainstream real-time instruments
 Fully customizable
Prep your PCR reaction mix  Load your plate  Run your real-time experiment!
No pipetting of individual primers!

Sample to Insight
miRNA expression profiling: miScript miRNA PCR Arrays (cont.)
‫‏‬miRBase Profiler miScript miRNA PCR Array
 Human
 Coverage through miRBase v21
 2402 primer assays!
 Mouse
 Coverage through miRBase v21
 1765 primer assays!
 Rat
 Dog
 Rhesus macaque
 Cow
 100% validated assays
 Each assay is bench validated
 Each array is quality controlled
 Leading miRNome coverage
 Completely scalable!
 Choose as many plates as you
want‫‏…‏‬profile‫‏‬the v21 miRNome
…‫‏‬profile‫‏‬only the v16 miRNome
 Contact product development if
there is interest in other species!
miRBase Profiler Arrays Benefits of miRBase Profiler Arrays
The most complete, validated miRNome available!

Sample to Insight
Are newly annotated miRNAs even expressed?
Liver tissue profiling of a pool of ten healthy male liver tissues
miScript
Vendor 2
Vendor 3

Sample to Insight
Limiting samples: miScript PreAMP Kit
‫‏‬miRNome profiling from as little as 1 ng total RNA
 Highly multiplex, PCR-based preamplification
 Compatible with all miScript miRNA PCR Arrays and miScript Primer Assays
 Enables miRNA profiling experiments using very limited amounts of starting material
 Cell or tissues: 1 ng total RNA
 Fluids:
 Serum / plasma: 50 µl or less
 Urine: Any amount
 CSF: Any amount
 Aqueous humor: Any amount
 When in doubt, ‘miScript PreAMP’ it!

Sample to Insight
High-throughput miRNA expression profiling: miScript Microfluidics
 First complete system for miRNA expression profiling on the Fluidigm® BioMarkTM
Why use miScript on the BioMark?
96 samples, 384 assays
4 Fluidigm Real-Time PCR Chips
5 h per Chip
36,864 data points in 20 h
(only 2 days!)

Sample to Insight
Agenda
31
Background1

Sample to Insight
Liver toxicity miRNA biomarker project with Dr. James Dear
Biomarker Discovery Workflow
Acetaminophen (APAP) overdose is a common poisoning
worldwide and can cause liver damage, potentially
resulting in acute liver failure and death
 In the US and UK, acetaminophen overdose is the most
common cause of acute liver failure
Scope of collaboration:
 Determine miRNA markers of acetaminophen poisoning
 Distinguish acetaminophen poisoning from other liver
syndromes
Experiment workflow: biomarker discovery
 Phase 1: 356 expressed miRNAs
 Narrowed list from 1809 miRNAs
 Phase 2: 85 differentially expressed miRNAs
 Phase 3: Classifier developed and tested that can
separate APAP-TOX from APAP-No TOX

Sample to Insight
Phase I: Qualitatively determine expressed miRNAs
Goal: Establish‫‏‬the‫“‏‬APAP‫‏‬miRNome”‫‏‬by‫‏‬assaying‫‏‬pooled‫‏‬samples‫‏‬with‫‏‬the‫‏‬human‫‏‬miRNome
Samples: 54 acetaminophen (APAP) overdose plasma samples
 27 APAP-no TOX: acetaminophen overdose without toxicity
 27 APAP-TOX: acetaminophen toxicity
Total RNA isolation: miRNeasy Serum/Plasma Kit
Random RNA pool preparation
 2 APAP-no TOX pools: 9 samples per pool
 2 APAP-TOX pools: 9 samples per pool
miRNA expression profiling: miScript PCR System
 Human miRNome V18 (1809 bench-verified primers)
Selection: Qualitative determination
 CT < limit of detection (CT = 35) with a single, sharp melt peak
How many miRNAs were selected? 356
 miRNAs expressed in all 4 pools: 132
 miRNAs expressed in 3 pools: 105
 miRNAs expressed in 2 pools: 114
 miRNAs expressed in 1 pool: 5

Sample to Insight
Phase II: Quantitatively determine differentially expressed miRNAs
Goal: Determine miRNAs differentially expressed in response to APAP toxicity
Total RNA Samples: 54 acetaminophen (APAP) overdose plasma samples from Phase I
 27 APAP-no TOX: acetaminophen overdose without toxicity
 27 APAP-TOX: acetaminophen toxicity
miRNA expression profiling: miScript PCR System
 356 human miRNAs
 Fluidigm BioMark HD (4 runs)
Results:
 85 differentially expressed miRNAs
 ± 3-fold, p-value < 0.05
 Well expressed in APAP-no TOX,
APAP-TOX, or both
 1 snoRNA
 7 invariantly expressed miRNAs
 Identified using NormFinder
 Very important for data normalization
miR-122-5p
miR-885-5p

Sample to Insight
Phase III: Develop and test classifier model
Goal: Develop and test a classifier that accurately separates APAP-TOX and APAP-no TOX
Step 1: Select the most predictive 16 miRNAs from training data set (93 targets)
 Train random forest classifier: 250 bootstrap subsamples (80%) without replacement
 Determined importance rank of each target
 Performed prediction on remaining 20% of samples
 Class separation magnitude and model error
 250 iterations of classifier training: Determined median importance rank of each target
 Selected the 16 most predictive targets
Step 2: Train a final random forest classifier using 16 most predictive miRNAs
Most predictive miRNAs
Importance Rank miRNA ID
1 - 11 miRNA #1 - #11
12 hsa-miR-122-5p
13 miRNA #13
14 hsa-miR-885-5p
15 miRNA #15
16 miRNA #16
Class Separation

Sample to Insight
Phase III: Develop and test classifier model (cont.)
Goal: Develop and test a classifier that accurately separates APAP-TOX and APAP-no TOX
Step 3: Test the random forest classifier on an independent, blinded test data set (81 samples)
 Sensitivity: 90%
 Specificity: 92%
 Classifier can efficiently separate
APAP-no TOX from APAP-TOX

Sample to Insight
Biomarker development
Important considerations
 Be novel! Initial whole miRNome screening = unique signatures!
If the group would have only profiled the first two plates (roughly 768 mature miRNAs):
 Only 64 of 92 differentially expressed or invariant miRNAs would have been assayed
 30% loss of data
 Two invariant miRNAs would have been missed
 Five miRNAs from optimal 16 miRNA signature would not have been assayed
 Three of top five miRNAs from the signature would not have been assayed
 CT value normalization is critical!
 snoRNAs / snRNAs do not exhibit robust expression in cell-free biofluids and should not be
selected as normalization controls
 Large assay panel normalization: CT mean of commonly expressed miRNAs
 Small assay panel normalization: Invariant miRNA(s)
 Verify miRNA signature on naïve samples
 Strong changes might be general indicators or even non-specific
 Other liver disease? Stress markers?
 Relatively weak changes might add specificity
 What’s next?
 Revisit literature, Ingenuity® Pathway Analysis (IPA), etc.
 Screen other types of samples to help define specificity and refine signature.

Sample to Insight
Circulating mRNA
Biomarker Discovery
 Exosomes contain stable RNA including mRNA
 Isolation
 exoRNeasy Serum / Plasma Maxi Kit
 Reverse Transcription/PreAMP
 RT2 PreAMP cDNA Synthesis Kit
 RT2 PreAMP Pathway Primer Mix
 Real-time PCR
 RT2 Profiler PCR Arrays
 RT2 qPCR Primer Assays

Sample to Insight
Agenda
39
Background1

Sample to Insight
Where can I find the products discussed today?
www.qiagen.com
www.qiagen.com/GeneGlobe

Sample to Insight
Sample to Insight miRNA portfolio
miRNeasy Mini Kit, miRNeasy Micro Kit miScript II RT Kit HiPerFect Transfection Reagent
miRNeasy 96 Kit miScript Plant RT Kit Attractene Transfection Reagent
miRNeasy FFPE Kit miScript PreAMP Kit miScript miRNA Mimics
miRNeasy Serum/Plasma Kit miScript SYBR Green PCR Kit miScript miRNA Inhibitors
Modified miRNeasy Mini Kit for plant
tissues
miScript miRNA PCR Arrays Custom miScript miRNA Mimics
PAXgene Tissue miRNA Kit miScript Microfluidics for Fluidigm Mimic and inhibitor controls
PAXgene Blood miRNA Kit miScript Primer Assay miScript Target Protector
Supplementary protocol for miRNA from
Plasma and Serum
miScript Precursor Assay
miScript miRNA Inhibitor 96 and 384
Plates and Sets
Profiling
QIAcube QIAgility Rotor-Gene QQIAGEN Service Core
FunctionalizationIsolation
Quantification
and profiling

Sample to Insight
Sample to Insight mRNA portfolio
Isolation
Quantification
and profiling
RNeasy Mini Kit RT2 First Strand cDNA Kits
RNeasy Microarray Tissue Mini Kit RT2 qPCR Master Mixes
RNeasy FFPE Kit RT2 Profiler PCR Arrays (Profiling)
RNeasy Micro Kit RT2 qPCR Primer Assays
PAXgene Blood RNA Kit GeneGlobe Data Analysis Center
QIAcube QIAgility Rotor-Gene QHigh-throughput

Sample to Insight
Thank you for attending today’s webinar!
Jonathan Shaffer, Ph.D.
Jonathan.Shaffer@qiagen.com
Contact QIAGEN
1-800-426-8157
BRCsupport@QIAGEN.com
Questions?

Biofluid miRNA profiling: from sample to biomarker: miRNA and its Role in Human Disease Webinar Series Part 1

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Biofluid miRNA profiling: from sample to biomarker: miRNA and its Role in Human Disease Webinar Series Part 1

Editor's Notes