Mi rna functional analysis 2013

Functional analysis of miRNA
Samuel J. Rulli, Jr. Ph.D.
samuel.rulli@QIAGEN.com

Sample & Assay Technologies

Topics for webinar
What is microRNA?
Manipulation of miRNA in cell lines using reporter
assays
miRNA Mimics
Inhibitors
Target protectors
Screening your target gene for miRNA regulation
Ready-to-use cDNA panels
Identification of miRNA regulation by qPCR
miRNA Targets PCR Arrays
Summary
Q & A session

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What Is Micro RNA?

RISC

Target mRNA

5’
CDS

3’

3’

5’
miRNA

Seed region

Endogenously expressed small functional RNAs (~ 21nt -24 nt)
Regulate mRNA expression post-transcriptionally
Involves the formation of RISC (RNA induced Silencing Complex)
Translational Repression and or mRNA degradation

Only partial sequence complementarity required
A given miRNA can regulate multiple mRNAs
A given mRNA can be regulated by multiple miRNAs
Another layer of complexity for regulating gene expression

Aberrant expression of miRNAs is associated with many diseases
e.g. Many different Cancers

-3-


Canonical pathway of microRNA 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 RNAse III-like
enzyme Drosha
Pre-miRNAs are then exported to the
Cytosol by Exportin 5
In the cytosol RNAse III-like 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

-4-


A growing picture of miRNA dysregulation in cancer,
developmental and metabolic pathways
In the last 5 years, a substantial number of studies and reviews have associated
the presence of various miRNAs with cell proliferation, resistance to apoptosis, and
differentiation in cancer cells.

.

.

.

For example:

.

Deletions of miRNA-regulated genes have been detected in more than 65%
of chronic lymphocytic leukemia cases, in 50% of mantle-cell lymphomas, in
16% to 40% of multiple myelomas, and in 60% of prostate cancers.
Other miRNA abnormalities have been reported in a wide variety of
Human neoplasms, including other hematologic malignancies such as
promyelocytic leukemia
Benign tumors such as leiomyoma and pituitary adenoma
Multiple types of carcinomas, including pancreatic, esophageal,
thyroid, lung, and breast
Neuroblastomas and glioblastomas

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Topics for webinar
What is microRNA?
assays
miRNA Mimics
Inhibitors
Target protectors
Summary
Q & A session

-6-


Manipulation of miRNA in cell lines
Functional interactions between miRNA and mRNA can be follow using:
Reporter assays: reporter gene has miRNA site in 3’ UTR which decreases
reporter gene translation or causes transcriptional degradation
Real-time PCR: monitors transcriptional degradation of mRNA
Western blot: monitors transcriptional or translational changes
Pathway reporter assay: monitors the pathway activity of specific transcription
Factors.

-7-


miRNA Mimics & Inhibitors: How do they work?

Transfection of
Mimics

Transfection of
Inhibitors

RISC

Target
Expression
Level

-8-



Transfection of
Mimics

Transfection of
Inhibitors

Mimics
increase
miRNA
activity

RISC

Target
Expression
Level

-9-


Comparable down-regulation by mimics and endogenous miRNAs

Hela cells with reporter assays for miR-1 or miR-16. miR-16 is endogenously
Expressed, while miR-1 was introduced using a miRNA mimic

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Stability of miRNA mimics

miRNA mimics are stable for at least 72 hours.
- 11 -


HDAC4 is a target of mir-1

HDAC4

HDAC4 had 2 miR-1 interaction sites. HeLa cells transfected with mir-1 show down
Regulation of HDAC4 at the mRNA and protein levels.


Transfection of
Mimics

Transfection of
Inhibitors

Inhibitor decrease
miRNA activity
RISC

Target
Expression
Level

- 13 -


mir-16 is decreased by miR-16 inhibitor in HeLa cells by reporter assay

- 14 -


Application Example: miRNA & Pathway Activity
The let-7 family of miRNAs is widely viewed as playing a role in tumor suppression.

.

Expression of let-7 family members is downregulated in many cancer types and during tumor
progression in comparison to normal tissue.

.

Humans have 10 mature let-7 family sequences (let-7a, 7b, 7c, 7d, 7e, 7f, 7g, 7i, miR-98, and
miR-202)

.

Interestingly, certain let-7 species are also known to be upregulated in some cancer types.
These data on the expression of let-7 in various cancers indicate that individual let-7 family
members can have different activities.

.

Question: Which let-7 family members effect cell cycle progression and
how?

.

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Cignal Reporters: What You Need

• Cignal Cell Cycle Pathway Reporter
Measures E2F/DP1 Activity

• Transfection of HEK293 cells

• Biological Test
•Let-7 family mimics

• Dual-Luciferase Assay

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Cignal Reporter Assays (45 Pathways)
Pathway
Amino Acid Deprivation
Androgen
Antioxidant Response
ATF6
C/EBP
cAMP/PKA
Cell Cycle
DNA Damage
EGR1
ER Stress
Estrogen
GATA
Glucocorticoid
Heat Shock Response
Heavy Metal Stress
Hedgehog
HNF4
Hypoxia
Interferon Regulation
Type I Interferon
Interferon Gamma
KLF4
Liver X

Transcription Factor
ATF4/ATF3/ATF2
Androgen Receptor
Nrf2 & Nrf1
ATF6
C/EBP
CREB
E2F/DP1
p53
EGR1
CBF/NF-Y/YY1
ER
GATA
GR
HSF
MTF1
GLI
HNF4
HIF-1
IRF-1
STAT1/STAT2
STAT1/STAT1
KLF4
LXR

- 17 -

Pathway
MAPK/ERK
MAPK/JNK
MEF2
c-myc
Nanog
NFκB
Notch
Oct4
Pax6
PI3K/AKT
PKC/Ca++
PPAR
Progesterone
Retinoic Acid
Retinoid X
Sox2
SP1
STAT3
TGFβ
Vitamin D
Wnt
Xenobiotic

Transcription Factor
Elk-1/SRF
AP-1
MEF2
Myc/Max
Nanog
NFκB
RBP-Jk
Oct4
Pax6
FOXO
NFAT
PPAR
PR
RAR
RXR
Sox2
SP1
STAT3
SMAD2/3/4
Vitamin D Receptor
TCF/LEF
AhR


Let-7 Family Members and the Cell Cycle

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Topics for webinar
What is microRNA?
assays
miRNA Mimics
Inhibitors
Target protectors
Summary
Q & A session

- 19 -


Approach to Identifying miRNA Regulators
miRNA mimic

miRNA

1

2

3

4

1) Treat Cells
2) Isolate RNA
3) First Strand cDNA Synthesis
4) qPCR for Gene Expression

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Transcriptome cDNA panels
Transcriptome PCR Arrays are ready-to-use cDNA panels for discovering the
miRNAs, that regulate the expression of any gene.

.

Each Transcriptome PCR Array is a panel of Transcriptomes or cDNAs:
90 Experimental cDNA samples
4 Vehicle Control cDNA samples
2 PCR Controls (only for SYBR green reactions)

.

Each experimental cDNA sample is derived from cells treated with a different
miRNA mimic treatment.
.

- 21 -


Example Transcriptome PCR Array

cDNA Sample 1
cDNA Sample 2
cDNA Sample 3
cDNA Sample 4
cDNA Sample 5
cDNA Sample 6

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Materials needed for miRNA discovery
.

.

.

Transcriptome PCR Array (1 for each GOI or HKG)
The Research Also Needs:
qPCR Thermocycler

Micropipettor
qPCR Master Mix
qPCR Assay (either SYBR® green or Probe-based Assays)
Computer with Microsoft Excel for Data Analysis

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SureFIND Protocol

- 24 -


miRNA pathways available

• Cancer miRNAs
• Immunopathology
• Development and differentiation
• Complete miRNome (~900 miRNAs)


Application Example: miRNA regulation of INPPL1
INPPL1, also known as SHIP2, is a SH2-containing 5'-inositol phosphatase

.

Converts PIP3 to PtdIns(3,4)P2, which negatively regulates PI3K signaling

.

Linked to Carcinogenesis

.

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Site Predictions for INPPL1

TargetScan

PicTar

miRANDA

miR-10

miR-184

miR-18a

miR-135

miR-205

miR-18b

miR-146

miR-342-3p

miR-184

miR-642

miR-18ab

miR-671-3p

miR-205
miR-27ab
miR-29abc

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Cancer miRNA Transcriptome
PCR Array Results for INPPL1

- 28 -


Site Predictions for INPPL1

TargetScan

PicTar

miRANDA

miR-10

miR-184

miR-18a

miR-135

miR-205

miR-18b

miR-146

miR-342-3p

miR-184

miR-642

miR-18ab

miR-671-3p

miR-205
miR-27ab
miR-29abc

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Compare Results with Research Community

What’s really exciting….
Three new miRNAs that play a role in INPPL1 expression
mir-193b
mir-138
mir-373

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Now what?............. Suggestions to Validate your “Hits”
miRNA-based Discovery
Perform a miScript miRNA mimic experiment
Identical Gain-Of-Function experiment
Perform a miScript miRNA inhibitor experiment
Loss-of-Function Experiment
Peform a miScript Target Protector experiment
ssRNA that disrupt a specific Target-miRNA interaction

.

- 31 -


Topics for webinar
What is microRNA?
assays
miRNA Mimics
Inhibitors
Target protectors
Summary
Q & A session

- 32 -


Identification of transcriptional degradation using qPCR:
RT2 Profiler microRNA Targets PCR Arrays

Transfection of
Mimics

Transfection of
Inhibitors

RISC

Target
Expression
Level

- 33 -


Anatomy of a RT2 Profiler microRNA Targets PCR Arrays
miR-21 Targets PCR Array
•

84 Experimentally verified or
predicted targets of miRNA

•

5 Housekeeping Genes

•

Genomic DNA Contamination
Control

•

Reverse Transcription
Controls (RTC) n=3

•

Positive PCR Controls
(PPC) n=3

Supported PCR Array Formats
•
•
•
•

96-Well Plate
384-Well Plate
100-Well Disc
96x96 Chip
- 34 -


Available RT² Profiler microRNA Targets PCR Arrays:
Human
miR-21
miR-155

Mouse
miR-21
miR-155

Rat
miR-21

miR-17 & miR-20a

miR-17 & miR-20a

miR-17 & miR-20a

miR-146a

miR-146a

miR-146a

miR-1 & miR-206

miR-1a & miR-206

miR-1 & miR-206

miR-34a

miR-34a

miR-34a

miR-221 & miR-222

miR-221 & miR-222

miR-221 & miR-222

let-7

let-7

let-7

Custom miRNA targets arrays are possible based on your miRNA of interest

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How RT2 Profiler PCR Arrays Work
cDNA Synthesis (C-03 kit)
– Genomic DNA Removal Step (5 min.)
– Reverse Transcription Step (20 min.)

.

Load Plates
1 Sample per PCR Array
2 minutes with multi-channel pipet

.

Run 40 cycle qPCR Program
Standard cycling conditions for all
Real Time PCR Instruments
2 hours

.

Upload and Analyze Data
15 minutes from Raw Ct to
Fold Change Data

.

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Clustergram
Data

.

.

.

Analysis

Web-Based Software
Scatter Plot
No installation or licensing needed
From Raw Ct Values to Fold Change Results
∆∆C(t) Method
Multiple Analysis Formats
Scatter Plot
Volcano Plot
Multi-Group Plot
Clustergram

Volcano Plot
Also available as downloadable Excel templates

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Summary

Global miRNA function can by manipulated by using mimics and inhibitors
Specific miRNA/mRNA interactions can be studied using target protectors
Pathway or transcription factor assays can be used to follow miRNA affects on
On cell biology
RT2 miRNA Targets PCR Arrays facilitate screening miRNA target genes using
verified qPCR Assays


Questions, Comments, Concern or Suggestions?

Answers to your technical and biological questions:
Technical Support:

888-503-3187

support@sabiosciences.com

Samuel Rulli
Samuel.Rulli@QIAGEN.com
Copy of slides? Type slides into questions box or fill out survey on exit
Want someone to call you? Type contact me into questions box
Want to try something? Request demos in the questions box.
miRNA mimics, inhibitors or target protectors
Cignal reporter assays
cDNA panels
miRNA Targets RT2 Profiler PCR Arrays

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Mi rna functional analysis 2013

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