quantification and profiling of mirnas

1 Sample & Assay Technologies

Isolation FunctionalizationQuantification

Quantification and Profiling of miRNAs:Challenges and Strategies

Subu Yerramilli, Ph.D.Associate Director R&[email protected]

The second in a series of QIAGEN webinars on miRNA 2011



QIAGEN miRNA Series1. Introduction to miRNABiogenesis and genomic organizationGeneration of miRNA diversity, regulation of expressionNormal function and mis-regulation in diseaseSample handling and profiling from FFPE and SerumIntroduction to quantification and functional genomics

Recorded version on QIAGEN website (coming soon)



QIAGEN miRNA Series2. Quantification and Profiling of miRNAs:Why quantify?Challenges and MethodologiesThe miScript SystemProfiling the miRNA-OmeData Analysis

Upcoming webinar (March 9th 2011):3. Small RNAs With Big Impact:

Functional Studies of miRNAs

Sample & Assay Technologies- 4 -

Why Quantify miRNA?

Virtually every publication included quantification experiments

Changes in miRNA correlated with gene expression changesin development, differentiation, signal transduction, infection, aging, and disease

Known miRNAs in Sanger DB miRNA publications in Medline

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miRNA Quantification Challenges

� Short sequences: ~16-26 nt

� Presence of miRNA isoforms� Near identical with ~1-3 nt differences� These differences can be any where in the sequence length� Potential for some degree of cross reactivity (Depends on the location of these differences)

� miRNAs with less than ideal GC% (very low or very high)

� Presence of repetitive sequences� hsa-miR-296-5p: AGGGCCCCCCCUCAAUCCUGU� hsa-miR-638: AGGGAUCGCGGGCGGGUGGCGGCCU� hsa-miR-940: AAGGCAGGGCCCCCGCUCCCC� mmu-miR-703: AAAACCUUCAGAAGGAAAGAA� mmu-miR-466b-5p: UAUGUGUGUGUACAUGUACAUA� mmu-miR-574-5p: UGAGUGUGUGUGUGUGAGUGUGU

� 3’-and 5’-end variations (Next slide)


3’-and 5’-End Variations in miRNA


miRNA Quantification Strategies

� Micro Array based approaches� Low specificity, needs further validation

� Expression analysis by deep sequencing� Expensive, and low throughput

� Real time PCR based approaches� Fast, flexible, scalable, accurate, sensitive


Real-Time PCR Based ApproachmiRNA 3’-End Dependent Reverse Transcription

e.g. hsa-Let-7a

5’-UGAGGUAGUAGGUUGUAUAGUU-3’

� Each miRNA detection requires a separate cDNA synthesis reaction

� A given RT primer could prime multiple miRNAs if these share the 3’-sequennces

� Doesn’t address 3’-end Polymorphism

TATCAA miRNA specific cDNA

www.mirbase.org


Real-Time PCR-Based ApproachesUniversal Reverse Transcription

5’-UGAGGUAGUAGGUUGUAUAGUU-3’

5’-UGAGGUAGUAGGUUGUAUAGUUAAAAAAAAAAAA-3’NVTTTTTTTTTTTT

qPCR withmiRNA specific FWD primerUniversal Reverse Primer

� A single cDNA prepared per sample is sufficient to interrogate many miRNAs

� 3’-end polymorphism tolerated

Poly A Polymerase

5’-UGAGGUAGUAGGUUGUAUAGUUAAAAAAAAAAAA-3’3’- ACTCCATCATCCAACATATCAATTTTTTTTTTTT

3’- ACTCCATCATCCAACATATCAATTTTTTTTTTTT

Reverse Transcriptase

Real Time PCR


miScript PCR System: Reverse Transcription & PCR

mRNA miRNA, other small RNAsmRNA miRNA, other small RNAs


miScript RT System: Linear cDNA Synthesis from 10 pg – 1µg RNA

� 10 pg to 1 µg of input miRNeasy RNA from HeLa S3 cells in miScript RT reaction (20 µl)� 1 pg to 100 ng cDNA used for each real time PCR

� A single cell is estimated to have ~10 pg of RNA

1 pg (<100 copies)


Quantification of miR-16 7-Log Dynamic Range in PCR


Specificity of miScript Primer Assays: Let-7 Isoforms

Human Let-7 Isoforms

Cross-reactivity Analysis

miRNA Sequence

--UGAGGUAGUAGGUUGUAUAGUU-- hsa-let-7a --UGAGGUAGUAGAUUGUAUAGUU-- hsa-let-7f --UGAGGUAGGAGGUUGUAUAGUU-- hsa-let-7e --UGAGGUAGUAGGUUGUGUGGUU-- hsa-let-7b --UGAGGUAGUAGGUUGUAUGGUU-- hsa-let-7c --AGAGGUAGUAGGUUGCAUAGUU-- hsa-let-7d --UGAGGUAGUAGUUUGUACAGUU-- hsa-let-7g --UGAGGUAGUAGUUUGUGCUGUU-- hsa-let-7i


Quantification of miRNA, mRNA and sn/snoRNA:From a Single cDNA Reaction

Template: 1 ng HeLa cDNA/PCR


Different 5 ′ & 3′ end extensions of Pre-miRNAs

Different 5 ′ & 3′ end extensions of Pre-miRNAs

miR-215

150125

100

75

M 1 10 10

Jurkat

HeLa

M 1 10 1 10

Jurkat

HeLa

miR-148b

150125

100

75

85100miR-148b

Bottom strand (3')

84147miR-215

Top strand (5')

Mature specific

Precursor specific

PCR Product size (Predicted; bp)

miRNA

85100miR-148b

Bottom strand (3')

84147miR-215

Top strand (5')

Mature specific

Precursor specific


miRNA

150125

100

75

M 1 10 1 10

Jurkat

HeLa

miR-345 miR-423

150125

100

75

M 1 10 1 10

Jurkat

HeLa

85105miR-423

Bottom strand (3')

84145miR-345

Top strand (5')

Mature specific

Precursor specific


miRNA

85105miR-423

Bottom strand (3')

84145miR-345

Top strand (5')

Mature specific

Precursor specific


miRNA

Gel analysis of miScript PCR products

miR-215miR-215

150125

100

75

150150125125

100100

7575

M 1 10 10

Jurkat

HeLa

M 1 10 1 10

Jurkat

HeLa

miR-148b

150125

100

75

M 1 10 1 10

Jurkat

HeLa

M 1 10 1 10

Jurkat

HeLa

miR-148b

150125

100

75

miR-148b

150125

100

75

150150125125

100100

7575

85100miR-148b

Bottom strand (3')

84147miR-215

Top strand (5')

Mature specific

Precursor specific


miRNA

85100miR-148b

Bottom strand (3')

84147miR-215

Top strand (5')

Mature specific

Precursor specific


miRNA

85100miR-148b

Bottom strand (3')

84147miR-215

Top strand (5')

Mature specific

Precursor specific


miRNA

85100miR-148b

Bottom strand (3')

84147miR-215

Top strand (5')

Mature specific

Precursor specific


miRNA

150125

100

75

M 1 10 1 10

Jurkat

HeLa

miR-345 miR-423

150125

100

75

M 1 10 1 10

Jurkat

HeLa

85105miR-423

Bottom strand (3')

84145miR-345

Top strand (5')

Mature specific

Precursor specific


miRNA

85105miR-423

Bottom strand (3')

84145miR-345

Top strand (5')

Mature specific

Precursor specific


miRNA150125

100

75

M 1 10 1 10

Jurkat

HeLa

miR-345 miR-423

150125

100

75

M 1 10 1 10

Jurkat

HeLa

150125

100

75

M 1 10 1 10

Jurkat

HeLa

miR-345

150125

100

75

M 1 10 1 10

Jurkat

HeLa

150125

100

75

150150125125

100100

7575

M 1 10 1 10

Jurkat

HeLa

M 1 10 1 10

Jurkat

HeLa

miR-345 miR-423

150125

100

75

M 1 10 1 10

Jurkat

HeLa

miR-423miR-423

150125

100

75

150150125125

100100

7575

M 1 10 1 10

Jurkat

HeLa

M 1 10 1 10

Jurkat

HeLa

85105miR-423

Bottom strand (3')

84145miR-345

Top strand (5')

Mature specific

Precursor specific


miRNA

85105miR-423

Bottom strand (3')

84145miR-345

Top strand (5')

Mature specific

Precursor specific


miRNA

85105miR-423

Bottom strand (3')

84145miR-345

Top strand (5')

Mature specific

Precursor specific


miRNA

85105miR-423

Bottom strand (3')

84145miR-345

Top strand (5')

Mature specific

Precursor specific


miRNA

Gel analysis of miScript PCR products

Only Mature miRNAs are Amplified:Pre-miRNAs are Not Co-Amplified


Data Normalization:Constant Expression Levels of Normalizer Non Coding RNAs

677769U91SCARNA17

6080U17A/B, E1SNORA73A

9303U25SNORD25

26826U6BRNU6B

26833U5ARNU5A

26869U1RNU1

GENE ID

ALSO KNOWN AS

OFFICIALSYMBOL

Expression of miScript control sn/snoRNAs: Human tissue Panel


QIAGEN Solutions for miRNA Research

Characterizing the miRNA- omeUsing the miScript System


How much RNA is Needed for miRNA Quantification?Depends on the Detection Limit of the System and miRNA Abundance

� System sensitivity: � miScript RT-PCR can detect 10 copies per PCR

� RNA abundance:� A ‘typical’ cell contains ~15-30 pg of total RNA� miRNA expression levels vary from as low as 10 copies/cell to

as high as 35,000 copies/cell

� How much template is needed to detect an abundant miRNA?� An abundant miRNA should be detectable with 10-15 pg of template


� What about miRNAs expressed at ~10-100 copies/cell?� Considering that 10x more template result in a 3.3 CT shift� Use 10-20 times more template (0.1 ng-0.2 ng) to detect rare miRNAs

� To be able to ‘call with certainty’ a miRNA is ‘not expressed’� use >20x more RNA (0.5 to 1.5 ng)

� As a guideline, consider using ~ 50 pg to 500 pg template/PCR� Up to 20,000 PCR reactions per 1 µg miScript RT reaction

� Desirable to use an RT system which tolerates a broad range of input RNA

� miScript allows 10 pg - 1µg in put RNA into RT reaction

How much RNA is Needed for miRNA Quantification?Depends on the Detection Limit of the System and miRNA Abundance


Expression of miRNAs in Jurkat cells

Total number of miRNAs tested : 328

Total number of miRNAs that show expression in Jurkat : 110

Total number of miRNAs showed >3.5 fold regulation: 30


Jurkat Cell Model System

Jurkat cells:� Acute T cell Leukemia cell line� Used as a model cell line for studying T-cell activation

PMA (Phorbol Myristyl Acetate)� Activates PKC which in turn activates NFkB & AP1

Ionomycin (Calcium Ionophore)� Activation of Ca2+/calmodulin-dependent signaling pathways,

which in turn activates NFAT

Jurkat cells

PMA PMA+ Ionomycin

One signal Two signalsNo signal

No Treatment


Activated Jurkat Cells:Differential Regulation of miRNAs

Measured at 24 hours, normalized to U6B ncRNA

miR-146a, miR-155 upregulated upon activation


3T3L1 Fibroblast Differentiation into Adipocytes

Preadipocyte Mature adipocyte

Nucleus

Lipid droplets

� Differentiation Induction by treatment with Insulin, Dexamethasone & Methylisobutylxanthine

Day -2 0 2 4 6 9 12 14

3T3L1 cells

Differentiation

Induction

InsulinNormal Growth medium

100%

Confluent

Differentiation

Induction


A Novel Two-Step miRNA Profiling Strategy

* up to n=10 samples used successfully

17730Conventional screening

60%% reduction

6912Two-step approach

Total # of PCRs

(n=10 Samples)

miRNA Profiling

Approach

RNA sample-1RNA Sample-2RNA Sample-3RNA Sample-n

cDNA-1cDNA-2cDNA-3cDNA-n

Pooled cDNA

miRNA profiling by miScript PCR system

miRNAs Expressed

miRNAs Not Expressed

miRNA profiling on individual samples

X

RNA sample-1RNA Sample-2RNA Sample-3RNA Sample-n

cDNA-1cDNA-2cDNA-3cDNA-n

Pooled cDNA

miRNA profiling by miScript PCR system

miRNAs Expressed

miRNAs Not Expressed

miRNA profiling on individual samples

X

****


Condensed miRNA Profiling by RT-PCRTracking miRNA Changes in 3T3-L1 Cells During Differentiation

cDNAPool

� What miRNAs are expressed in resting, proliferative and differentiated Adipocytes?

� Profiling using 591 miScript primer assays (miRBase v12, Mouse)

Total RNA

miScript RT

Day -2 0 2 4 6 9 12 14

Differentiation

Induction

miRNAs Expressed = 192 (32%)No expression = 399

192 miRNAs Expressed

399 miRNAs Not expressed

192 miRNAs Expressed

399 miRNAs Not expressed


Relative Expression of Selected Mature and pre-miRNAsNIH 3T3L1: Day 1 vs. Day 9

General trend is that change in mature miRNA correlates with pre-miRNAbut there are exceptions which point to differences in processing efficiency/turnover

67 miRNAs Differentially Regulated125 miRNAs Unchanged

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Mature PrecursorMature Precursor


qRT-PCR Data Analysis

� Absolute quantification� Absolute input copies, based on a standard curve

� Relative quantification� Comparative CT method

(also known as the 2-∆∆CT method)

� Selection of internal control

� Selection of calibrator

(e.g. untreated control or normal sample)

� Assumes that the PCR efficiency of the target gene is similar to the internal control gene (and that the efficiency of the PCR is close to 1)

� Fold change = 2-∆∆CT

= [{gene of interest (CT) – internal control (CT) sample A} –

{gene of interest (CT) - internal control (CT) sample B}]

(1) Schmittgen TD, Livak KJ.(2008):Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc.;3(6):1101-8

(2) Livak, KJ, and Schmittgen, TD.(2001): Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2-∆∆CT Method METHODS 25, 402–408

(3) www.Gene-Quantification.info

CT = 23.8


� cDNA Synthesis � 2 hours

� Load Plates � 2 minutes

� Run 40 cycle qPCR Program � 2 hours

� Upload and Analyze Data� 15 minutes

How RT2 miRNA PCR Arrays Work


RT2 Data Analysis

� Web-Based Software� No installation needed

� From Raw Ct Values to Fold Change Results� Using ∆∆C(t) Method

� Multiple Analysis Formats� Scatter Plot� Volcano Plot

� Multi-Group Plot

� Clustergram

Also available as downloadable Excel templates


RT2 Data Analysis





� Clustergram


Scatter Plot


RT2 Data Analysis





� Clustergram


Scatter Plot

Volcano Plot


RT2 Data Analysis





� Clustergram


Scatter Plot

Volcano Plot

Clustergram

Sample & Assay Technologies

RT2-miRNA PCR ArraysGenome-Wide, Disease, & Pathway-Focused Analysis

Human, Mouse & Rat miRNA-Ome

Human & Mouse miFinder88 abundantly expressed & well-characterized miRNAs

.Human, Mouse & Rat Cancer

.Human, Mouse & Rat Cell Development & Differentiation

Human, Mouse & Rat Immunopathology

Human, Mouse & Rat Inflammation

Human, Mouse, and Rat Neurological Dev and Disease

Human, Mouse, and Rat Brain Cancer

Human Disease Circulating in Serum


miScript System:Pre-miRNA Quantification

Quantitect mRNA Assays

mRNAs miRNAs

Other small RNAs

Pre-miRNAs

Pre-miRNA specificFwd & Rev Primers

miScript Primer Assays

miScript Primer AssaysFor non-coding RNA

miScriptSystem


Pre-miRNA Stem-Loop Quantification

miScript miRNA assays detect mature miRNAs only Mature specific Fwd & Universal Rev primerShould not co-amplify Pre-miRNA

miScript Pre-miRNA assays amplify precursors onlypre-miR specific Fwd & Rev Primers Should not co-amplify mature miRNA

.Pre-miRNA assays amplify all RNA species that conta in the stem loop

Pri-miRNAsPre-miRNAs

� Study transcriptional and/or post-transcriptional r egulation in miRNA biogenesis, and downstream implications

� Definitively identify which closely related genomic loci are expressed


Multiple Precursors Can Generate the Same Mature miRNA : e.g. hsa-mir-7

hsa-mir-7-1, Intronic of HNRNPK, CHR-9

hsa-mir-7-2, Intergenic, CHR-15

hsa-mir-7-3, Intronic of C19orf30, CHR-19

hsa-mir-7UGGAAGACUAGUGAUUUUGUUGU


miScript Precursor Assays Distinguish Pre-miRNA Stem-Loop Paralogs

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Pre-m

ir-7-

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Pre-m

ir-7-

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ir-7-

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iR-7

U6B

SNORA73A

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g H g H g H M

255075

100125

g = gDNAH = HeLa cDNA

miR

-7-1

miR

-7-2

miR

-7-3

bp

77miR-7-3

55miR-7-2

76miR-7-1

Expected Size (bp)

PCR product

77miR-7-3

55miR-7-2

76miR-7-1

Expected Size (bp)

PCR product

Only Pre-miR-7-1 stem loop shows detectable expres sion in HeLa S3 cells

10 ng cDNA/PCR


Expression of Let-7a Precursors in Different Tissues

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xpre

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Pre-Let-7a-2

Pre-Let-7a-3

Relative Expression = 2 -∆CT X 105

Normalizer Used: U6B10 ng cDNA /PCR


Expression of miR-18a and its PrecursorA member of the miR-17-92 Cluster (13q31.3)

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∆CT Pre-miR-18a

∆CT mature-miR-18a

∆C

T (

Rel

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U6B

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gDN

AAdipose Brain Heart Liver Ovary HeLa L

P M P M P M P M P M P M P 125 bp

75 bp

50 bp

P) Pre-miR-18a specific M) Mature miR-18a specific

Guil, S & Ca´ceres, JF., (2007) Nat Struct Mol Biol;14(7):591-6.

Pre-miR-18a Specific Product

Mature miR-18a Specific Product


miScript System

Circulating miRNA in Serum and Plasma


Workflow for Quantification of Circulating miRNA

Blood collection & plasma/serum

isolation

RNA Prep

+ Cel-miRNAs

cDNA synthesis

Real-time PCR

Data analysis &

normalization

QIAzol Bind Wash Elute

AAAAAAA

TTT

AAAAAAATTT

miRNeasy

Serum & Plasma Protocol

miScript RT

RT2-miRNA RT

miScript (single miRNA Assays)

RT2 miRNA PCR Arrays

Data analysis tools – FREE!


Circulating miRNA: Special Considerations

� Serum or Plasma? Be consistent in collection and handling.

� Take steps to insure minimum cell lysis or carryover of cells

� Use an exogenously spiked in normalizer (e.g. C. elegans spike in)

� No ‘normal’ levels of any RNA established

� Platelets may contain miRNAs (Landry P et al 2009)

� For plasma

� Do NOT use heparin as anticoagulant as it can interfere in RTPCR

� Appropriate caveats in quantifying circulating miRNAs as markers

� Use an optimized isolation protocol (e.g. miRNeasy Plasma protocol)

Landry, P., et. al.,(2009) Existence of a microRNA pathway in anucleate platelets. Nat Struct Mol Biol 16:961


Summary

� Challenges in miRNA quantification

� miRNA quantification by real time PCR

� Available tools for miRNA profiling experiments

� Critical aspects of template requirements

� Examples of miRNA profiling studies

� Precursor detection� Measuring the relative levels of mature miRNA and its precursor� Identifying the genetic locus responsible for a given miRNA expression in a cell

� RT2 miRNA arrays for simple and easy miRNA Profiling

� Data analysis

� Serum miRNA quantification


www.GeneGlobe.com


Thank you!

quantification and profiling of mirnas

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