Introduction to microRNA:

miRNA Function, Profiling and Data Analysis

Ali Bierly, Ph.D.

allison.bierly@qiagen.com

Sample to Insight

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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
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Sample to Insight
Meeting the challenges of miRNA research

Agenda

miRNA background

Sample prep

Real-time PCR assays

Data analysis

Interpretation

Sample to Insight

miRNAs: master regulators of gene expression

Small but mighty!
miRNAs are 21-nucleotide small non-coding RNAs that are expressed in virtually all tissues
Changes in miRNA expression can be correlated with gene expression changes in development,
differentiation, signal transduction, infection, aging and disease
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
Meeting the challenges of miRNA research

How do miRNAs interact with mRNAs?

Basis of miRNAmRNA interaction
Seed region: nucleotides 27 in 5 region of miRNA
Most evolutionary conserved miRNA region
Most frequently complementary to target 3-UTRs
Often sufficient to confer mRNA recognition
Beyond the seed region
3 end also contributes (extensive pairing is rare)
Some cases: central 1112 continuous base pairs
Result of interaction
Suppression of gene expression
Rare cases: increase gene expression
References
Grimson, A., et al, Mol. Cell 2007, 27, 91105
Image from Bartel, D.P., Cell 2009, 136, 215233
Guo, H., et al, Nature 2010, 466, 835840
Thomson, D.W., et al, Nucleic Acids Res 2011, 19
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Meeting the challenges of miRNA research

How do you determine miRNAmRNA interactions?

Step 1: Prediction algorithms!
Target Prediction is based on:
Bioinformatics

Pitfalls of using prediction algorithms:

Large number of candidate mRNAs for a given
miRNA
May not incorporate all miRNA targeting
possibilities
Different algorithms produce different target lists
Potential for false positive rate of prediction

Seed region match

Position in 3 UTR
Cross-species conservation
Central sequence homology

Wet-lab research

Empirical evidence from microarrays

Reporter systems
Prediction Algorithm

Website

TargetScan

http://www.targetscan.org/

Pictar

http://pictar.mdc-berlin.de/

MicroCosm Targets

http://www.ebi.ac.uk/enright-srv/microcosm/htdocs/targets/v5/

DIANA

http://diana.cslab.ece.ntua.gr/microT/

miRANDA

http://www.microrna.org/microrna/home.do

TarBase (experimentally
supported)

http://diana.cslab.ece.ntua.gr/tarbase/

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Meeting the challenges of miRNA research

How do you determine miRNAmRNA interactions?

Step 2: Experimental techniques!

miRNA target screening

Gene expression analysis (inferred targets)

RNAseq
Microarrays
qPCR
Immunoprecipitation (direct targets)
HITS-CLIP
PAR-CLIP
Biotin tagged miRNA

Gene-specific validation

qPCR
Luciferase reporter assays
Western blot
5 rapid identification of cDNA ends (5 RLM-RACE)

Image from Chi, S.W. et al. (2009) Nature 13, 479.

Sample to Insight
Meeting the challenges of miRNA research

What is the role of miRNA in human disease?

Sample to Insight
Meeting the challenges of miRNA research

Potential events that disrupt normal miRNA activity

Disruption of miRNAmRNA interaction

Altered transcription

Genomic instability

Methylation
Histone modification
Transcription factor

Amplification/deletion
Translocation
Insertional mutagenesis

Drosha processing

Dicer processing

Loss of miRNA
binding site in target
SNP or mutation
Alternative splicing
Loss/change of 3-UTR
Sample to Insight
Meeting the challenges of miRNA research

Unique signatures in human cancer

miRNAs located in genomic regions amplified in cancers (e.g.

miR-17-92 cluster) can function as oncogenes, whereas miRNAs
located in portions of chromosomes deleted in cancers (e.g.
miR-15a-miR-16-1 cluster) can function as tumor suppressors
Abnormal expression of miRNAs has been found in both solid
and hematopoietic tumors
miRNA expression fingerprints correlate with clinical and
biological characteristics of tumors including tissue type,
differentiation, aggression and response to therapy

In the last 15 years, a substantial number of studies and reviews have

associated the presence of various miRNAs with cell proliferation,
resistance to apoptosis, invasiveness and differentiation in cancer cells

Sample to Insight
Meeting the challenges of miRNA research

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QIAGEN Sample to Insight solutions for miRNA research

QIAgility
RotorGene Q
Compatibility with all
Real-time PCR instruments

Instruments

QIAcube

Real-time
PCR assays

Sample prep

Kits/
solutions

miRNeasy Mini
miRNeasy
Micro
miRNeasy
FFPE
miRNeasy
Serum/Plasma
ExoRNeasy
Serum/Plasma

miScript PCR System

miScript PreAMP
miScript Microfluidics
miScript PCR Arrays
miScript Primer
Assays

Data analysis

GeneGlobe
Data Analysis
Center

Interpretation

Ingenuity Pathway
Analysis
miScript Mimics
miScript Inhibitors

Sample to Insight

Webinar 4: Functional Analysis of miRNA

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Agenda

miRNA background

Sample prep

Real-time PCR assays

Data analysis

Interpretation

Sample to Insight

12

QIAGEN Sample to Insight solutions for miRNA research

QIAgility
RotorGene Q
Compatibility with all
Real-time PCR instruments

Instruments

QIAcube

Real-time
PCR assays

Sample prep

Kits/
solutions

miRNeasy Mini
miRNeasy
Micro
miRNeasy
FFPE
miRNeasy
Serum/Plasma
ExoRNeasy
Serum/Plasma

miScript PCR System

miScript PreAMP
miScript Microfluidics
miScript PCR Arrays
miScript Primer
Assays

Data analysis

GeneGlobe
Data Analysis
Center

Interpretation

Ingenuity
Pathway Analysis
miScript Mimics
miScript
Inhibitors

Sample to Insight

Webinar 4: Functional Analysis of miRNA

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Sample to Insight: sample prep

A total RNA solution for every sample type
Cells, fresh tissue, frozen tissue
miRNeasy Mini Kit
miRNeasy Micro Kit
miRNeasy 96 Kit
FFPE tissue
miRNeasy FFPE Kit
Fluids (serum, plasma, urine, CSF, saliva etc.)
miRNeasy Serum / Plasma Kit
Exosome enrichment/isolation from serum/plasma
ExoRNeasy Serum/Plasma Maxi Kit

Sample to Insight
Meeting the challenges of miRNA research

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Agenda

miRNA background

Sample prep

Real-time PCR assays

Data analysis

Interpretation

Sample to Insight

15

QIAGEN Sample to Insight solutions for miRNA research

QIAgility
RotorGene Q
Compatibility with all
Real-time PCR instruments

Instruments

QIAcube

Real-time
PCR assays

Sample Prep

Kits/
solutions

miRNeasy Mini
miRNeasy
Micro
miRNeasy
FFPE
miRNeasy
Serum/Plasma
ExoRNeasy
Serum/Plasma

miScript PCR System

miScript PreAMP
miScript Microfluidics
miScript PCR Arrays
miScript Primer
Assays

Data analysis

GeneGlobe
Data Analysis
Center

Interpretation

Ingenuity
Pathway Analysis
miScript Mimics
miScript
Inhibitors

Sample to Insight

Webinar 4: Functional Analysis of miRNA

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miScript
Complete miRNA quantification system

Reverse transcription
miScript II RT Kit
Preamplification for limiting RNA amounts
miScript PreAMP PCR Kit
miScript PreAMP Primer Mixes
Single cell miRNA quantification
miScript Single Cell qPCR System
High-throughput expression analysis
miScript miRNA PCR Arrays
Low-throughput miRNA quantification
miScript Primer Assays
Real-time PCR reagents
miScript SYBR Green PCR Kit
miScript Microfluidics PCR Kit (for Fluidigm
Biomark HD)

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Meeting the challenges of miRNA research

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miScript PCR System

Basic workflow
1. Isolate total RNA
2. Perform reverse-transcription
3. Perform PreAMP (optional)
4. Prepare PCR pre-mix
5. Load PCR arrays or plates
6. Perform real-time PCR
7. Analyze data

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Meeting the challenges of miRNA research

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Preamplification for limiting samples: miScript PreAMP Kit

miRNome profiling from as little as 1 ng total RNA or biofluids with limited 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 l or less
Urine: Any amount
CSF: Any amount
Aqueous humor: Any amount
When in doubt, miScript PreAMP it!
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Meeting the challenges of miRNA research

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miScript Single Cell qPCR Kit: Product Overview

What is the product?

Lysis, cDNA synthesis and preamplification reagents to enable universal mature

miRNA quantification from individual cells.

Whats the required starting material?

1-100 cells

25 pg to 1 ng of purified RNA (a single-cell RNA amount)

Should animal or plant samples be used?

Animal samples

If you have inquiries about plant samples, please direct them to our microRNA
R&D expert Jonathan Shaffer (jonathan.shaffer@qiagen.com)

What miScript products are compatible with the miScript Single Cell qPCR Kit?

All animal miScript Primer Assays

All animal miScript miRNA PCR Arrays

miScript SYBR Green PCR Kits

miScript Microfluidics PCR Kit

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Title, Location, Date

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High-throughput expression analysis: miScript PCR Arrays

What are miScript PCR Arrays? Wet-lab verified miRNA primer assays pre-dried in PCR plates

miRNome arrays
Most species
Broadest content
Targeted miRNome arrays
sub-miRNomes
Focused arrays
Biological pathways and diseases
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!
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Meeting the challenges of miRNA research

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Anatomy of a miScript miRNA PCR Array

96-well format: 84 miRNA + 12 controls

84 mature miRNAs

cel-miR-39

SNORD61; SNORD68; SNORD72

SNORD95; SNORD96A; RNU6-2

miRTC

PPC

Spike in
control

miScript PCR controls for

normalization

RT
control

PCR
control

cel-miR-39

Alternative data normalization using exogenously spiked Syn-cel-miR-39 miScript miRNA Mimic

miScript PCR controls

miRNA reverse transcription control (miRTC)

Data normalization using the CT method of relative quantification

Assessment of reverse transcription performance

Positive PCR control (PPC)

Assessment of PCR performance

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Meeting the challenges of miRNA research

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PCR arrays or individual miScript Primer Assays?

What option is best for your experiment?

Its a balance of samples and assays

miRNome screening: always PCR arrays
More than 24 assays: always PCR arrays
Less than 24 assays: depends on the number of samples
Limited samples (16 or less): individual PCR assays
Extensive number of samples (16, 50, 100, etc.): PCR arrays

or

Sample to Insight
Meeting the challenges of miRNA research

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miScript PCR System performance

Exceptional linearity and specificity

Detection of 10 copies
to >106 copies of miRNA

Linearity of six logs of input RNA

32

40

miR-21

miR-16

28

miR-21
Linear (miR-16)

30

Linear (miR-20a)
Linear (miR-21)

Mean CT

Mean CT

Linear (miR-21)

miR-20a

35

25
20
15

24

20

16

10
-2

-1

Log (ng) of RNA in cDNA synthesis using the HiFlex Buffer

12
1

Log copy number of miRNA using the HiFlex Buffer

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Meeting the challenges of miRNA research

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miScript PCR System: exceptional specificity

Excellent discrimination between closely related miRNA family members

Relative detection (as % of perfect match)

cDNA
used in
PCR

Let-7b

Let-7c

miR-98

Let-7d

Let-7e

Let-7a

Let-7f

Let-7g

Let-7i

Let-7b

100.0

1.8

0.0

0.0

0.0

0.0

0.0

0.0

0.0

Let-7c

0.5

100.0

0.0

0.0

1.0

0.1

0.0

0.0

0.0

miR-98

0.0

0.2

100.0

0.1

0.0

0.1

0.0

0.0

0.1

Let-7d

0.1

0.0

0.0

100.0

0.0

0.4

0.0

0.0

0.0

Let-7e

0.1

0.0

0.0

0.0

100.0

0.2

0.0

0.0

0.0

Let-7a

0.1

0.6

0.0

0.5

3.9

100.0

0.1

0.0

0.0

Let-7f

0.6

0.1

0.0

0.1

0.0

1.1

100.0

0.1

0.1

Let-7g

0.6

0.2

0.0

0.1

0.0

0.0

0.0

100.0

0.2

Let-7i

0.1

0.0

0.0

0.0

0.0

0.0

0.0

0.1

100.0

miScript Primer Assay used

Sample to Insight
Meeting the challenges of miRNA research

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Focused miRNA expression profiling

Normal lung

36

36

32

32

28

28

24

24

20
16
12

FFPE Isolation 1

FFPE Isolation 2
FFPE Isolation 3

4
1

7 13 19 25 31 37 43 49 55 61 67 73

Lung tumor

40

CT Value

CT Value

40

20
16
12

FFPE Isolation 1

FFPE Isolation 2
FFPE Isolation 3

4
1

7 13 19 25 31 37 43 49 55 61 67 73

One 5 m FFPE section used per FFPE isolation

Each isolation is from a different section
On average, each isolation provided enough total RNA for:
Two full human miRNome profiles
Ten pathway-focused PCR arrays

RT: 125 ng total RNA, HiSpec Buffer

qPCR: Human miFinder miScript miRNA PCR Array (0.5 ng cDNA per well)
Sample to Insight
Meeting the challenges of miRNA research

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Focused miRNA expression profiling, continued

FFPE samples

Sample to Insight
Title, Location, Date

27

High Content (HC) microRNA expression profiling

Serum Sample

Sample to Insight
Title, Location, Date

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miScript PreAMP: Excellent result, a fraction of the input

Sample to Insight
Title, Location, Date

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QIAGEN Sample to Insight solutions for miRNA research

QIAgility
RotorGene Q
Compatibility with all
Real-time PCR instruments

Instruments

QIAcube

Real-time
PCR assays

Sample prep

Kits/
solutions

miRNeasy Mini
miRNeasy Micro
miRNeasy FFPE
miRNeasy
Serum / Plasma
ExoRNeasy
Serum / Plasma

miScript PCR System

miScript PreAMP
miScript Microfluidics
miScript PCR Arrays
miScript Primer Assays

Data analysis

GeneGlobe
Data Analysis
Center

Interpretation

Ingenuity Pathway
Analysis
miScript Mimics
miScript Inhibitors

Sample to Insight
Webinar 4: Functional Analysis of miRNA

30

Real-time 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 100%)
Fold change = 2-CT
CT = 23.8

CT = CTGene - CTNormalizer
CT = CT (sample 2) CT (sample 1) where sample 1
is the control sample and sample 2 is the experimental
sample

(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, 402408

(3)

www.Gene-Quantification.info

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Meeting the challenges of miRNA research

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Data analysis workflow

Steps 1 and 2:
Set baseline and threshold to determine CT values

Step 3:
Export CT values

Step 4:
Analyze data using CT method of relative quantification

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Meeting the challenges of miRNA research

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Data analysis step 1: Set your baseline

Baseline
Definition: Noise level in early cycles where there is no detectable increase in
fluorescence due to PCR products
How to set:

Observe amplification plot using the Linear View

Determine the earliest visible amplification
Set the baseline from cycle 2 (or 3) to two cycles before the earliest visible amplification
Note: The number of cycles used to calculate the baseline can be changed and should be
reduced if high template amounts are used

Important: Ensure baseline settings are

the same across all PCR runs in the
same analysis to allow comparison of
results

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Meeting the challenges of miRNA research

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Data analysis step 2: Set your threshold

Threshold
Purpose: Used to determine the CT (threshold cycle) value. The point at which
the amplification curve intersects with the threshold line is called the CT
How to set:
Observe amplification plot using the Log View
Place the threshold in the lower half of the log-linear range of the amplification plot, above
the background signal
Note: Never set the threshold in the plateau phase

Important: Ensure threshold settings

are the same across all PCR runs in
the same analysis to allow
comparison of results

Sample to Insight
Meeting the challenges of miRNA research

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Data analysis Step 3: Export Ct values

Normal lung

36

36

32

32

28

28

24

24

20
16
12

FFPE Isolation 1

FFPE Isolation 2
FFPE Isolation 3

4
1

7 13 19 25 31 37 43 49 55 61 67 73

Lung tumor

40

CT Value

CT Value

40

20
16
12

FFPE Isolation 1

FFPE Isolation 2
FFPE Isolation 3

4
1

7 13 19 25 31 37 43 49 55 61 67 73

One 5 m FFPE section used per FFPE isolation

Each isolation is from a different section
On average, each isolation provided enough total RNA for:
Two full human miRNome profiles
Ten pathway-focused PCR arrays

RT: 125 ng total RNA, HiSpec Buffer

qPCR: Human miFinder miScript miRNA PCR Array (0.5 ng cDNA per well)
Sample to Insight
Title, Location, Date

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Data analysis step 4: Analyze data

CT method of relative quantification

Normal (N) lung total RNA

N cDNA (Iso. 1)

N cDNA (Iso. 2)

Lung tumor (T) total RNA

N cDNA (Iso. 3)

Exported CT values

CT = CTmiRNA AVG CTSN1/2/3/4/5/6

T cDNA (Iso. 1)

Calculate CT
for each miRNA
on each array

T cDNA (Iso. 2)

T cDNA (Iso. 3)

Exported CT values

CT = CTmiRNA AVG CTSN1/2/3/4/5/6

Tip for choosing an appropriate snoRNA / snRNA controls for normalization

Make sure that the selected controls are not influenced by the experimental conditions
Sample to Insight
Meeting the challenges of miRNA research

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Data analysis step 4: Analyze data (cont.)

CT method of relative quantification

Normal (N) lung

CT

Lung tumor (T)

CT

CT

Calculate CT
for each miRNA
on each array

CT

CT

CT

Calculate average
CT for each miRNA
within group (N or T)

CT + CT + CT

CT + CT + CT

Calculate CT for
each miRNA
between groups
(T N)

CT = Avg. CT (T) Avg. CT (N)

Calculate fold-change for each miRNA (T vs. N)

2-(CT)
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Data analysis example 1

Increased expression in a tumor sample
Two conditions: Normal and Tumor
miRNA (hsa-miR-21-5p)
Normal CT = 21
Tumor CT = 15

Normalizer (RNU6-2)
Normal CT = 16
Tumor CT = 14

Analysis
1. Calculate CT for each condition (i.e., normalize your miRNA CT values)

2.

Calculate CT (tumor relative to normal) using the equation CT (T) CT (N)

3.

CT (tumor relative to normal): 1 5 = 4

Calculate fold-change (tumor relative to normal) using the equation 2-CT

4.

Normal: 21 16 = 5
Tumor: 15 14 = 1

2-CT (tumor relative to normal): 2-(-4) = 16

Calculate fold-regulation

If the fold-change is greater than 1, the result may be reported as a fold upregulation

Compared to the normal sample, hsa-miR-21-5p is 16-fold

upregulated in the tumor sample
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Data analysis example 2

Decreased expression in a tumor sample
Two conditions: Normal and Tumor
miRNA (hsa-miR-16-5p)
Normal CT = 15
Tumor CT = 16

Normalizer (RNU6-2)
Normal CT = 16
Tumor CT = 14

Analysis
1. Calculate CT for each condition (i.e. normalize your miRNA CT values)

2.

Calculate CT (tumor relative to normal) using the equation CT (T) CT (N)

3.

CT (tumor relative to normal): 2 (1) = 3

Calculate fold-change (tumor relative to normal) using the equation 2-CT

4.

Normal: 15 16 = 1
Tumor: 16 14 = 2

2-CT (tumor relative to normal): 2(3) = 0.125

Calculate fold-regulation:

If the fold-change is less than 1, the negative inverse of the result may be reported as a fold
downregulation (1 / 0.125 = 8)

Compared to the normal sample, hsa-miR-16-5p is 8-fold

downregulated in the tumor sample
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Meeting the challenges of miRNA research

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miScripts straightforward data analysis solution

Incorporating the free GeneGlobe Data Analysis Center

Steps 1 and 2:
Set baseline and threshold to determine CT values

Step 3:
Export CT values

Step 4:
Access the free data analysis software at
www.qiagen.com/GeneGlobe

Step 5 and on:

Automatic data using CT method of relative quantification

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Data analysis step 5: GeneGlobe Data Analysis Center

Analyze your miScript miRNA PCR Array and miScript Primer Assay results!

Web-based software
No installation needed
Tailored for each array

Raw CT values to results

Using CT Method

Multiple analysis formats

Scatter plot
Volcano plot
Multi-group plot
Clustergram

Sample to Insight

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QIAGEN Sample to Insight solutions for miRNA research

QIAgility
RotorGene Q
Compatibility with all
Real-time PCR instruments

Instruments

QIAcube

Real-time
PCR assays

Sample prep

Kits/
solutions

miRNeasy Mini
miRNeasy Micro
miRNeasy FFPE
miRNeasy
Serum / Plasma
ExoRNeasy
Serum / Plasma

miScript PCR System

miScript PreAMP
miScript Microfluidics
miScript PCR Arrays
miScript Primer Assays

Data analysis

GeneGlobe
Data Analysis
Center

Interpretation

Ingenuity Pathway
Analysis
miScript Mimics
miScript Inhibitors

Sample to Insight

42

Ingenuity Pathway Analysis (IPA)

Asking whats next? by modeling, analyzing and understanding complex 'omics data
Analysis of gene expression / miRNA / SNP microarray data
Deeper understanding of metabolomics, proteomics and RNAseq data
Identification of upstream regulators
Insight into molecular and chemical interactions and cellular phenotypes
Discoveries about disease processes

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Testing whats next?

Manipulating miRNA function

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Where can I find the products discussed today?

www.qiagen.com
www.qiagen.com/GeneGlobe

Sample to Insight

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Introduction to microRNA
What we have covered today

miRNAs are master regulators of gene expression

QIAGEN has a Sample to Insight solution specifically tailored for you!

Sample prep

Real-time PCR assays

Data analysis

Interpretation

Choose QIAGEN and turn your hypotheses into actionable insights!

Sample to Insight

46

Questions?
Thank you for attending todays webinar!

Allison Bierly, Ph.D.

Allison.bierly@qiagen.com

Contact QIAGEN
1-800-426-8157
BRCsupport@QIAGEN.com

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