This slideshow highlights the use of miRNA mimics, inhibitors and target protectors to increase, decrease and adjust the cellular concentration of miRNA and disrupt specific miRNA–mRNA interactions. A ready-to-use screening tool for identifying miRNA targets and info on how to predict mRNA targets using miRNA expression data are also highlighted.

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Functional analysis of miRNA
Martin Kreutz
Martin.Kreutz@qiagen.com
Scientist, Product Development

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Legal disclaimer
Webinar 4: Functional Analysisof miRNA 2
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.

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Welcome to our four-part webinar series on miRNAs
Webinar 4: Functional Analysisof miRNA 3
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

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miScript miRNA Target Protectors4
Agenda
miRNA background1
miScript miRNA Mimics2
miScript miRNA Inhibitors3
4
Controls5

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 Changes in miRNA can be
correlated with gene expression
changes in development,
differentiation, signal transduction,
infection, aging and disease
 After expression analysis of
miRNAs, functional studies are the
next step to understand the meaning
of the observed changes in
expression
 As a single miRNA can have a large
group of potential targets. Functional
analysis is a valuable tool to
investigate the involvement of a
differentially regulated miRNA in the
process of interest
miRBase Entries
Webinar 4: Functional Analysisof miRNA
Why assess the function of microRNAs (miRNAs)?

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6Webinar 4: Functional Analysisof miRNA
Canonical pathway of miRNA biogenesis
 Transcribedby RNApolymerase 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 inRISC
 miRNAs with high homology to the target mRNA
lead to mRNA cleavage
 miRNAs with imperfect base pairing to the target
mRNA lead to translational repressionand / or
mRNA degradation

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Mechanisms of gene silencing by miRNA:
 Ribosome drop-off
 Deadenylation and degradation
 miRNA sequestration
Translational Repression
and/or
Transcript Degradation
P Body Sequestration
Translational Repression
and/or
Transcript Degradation
P Body Sequestration
Guo, H et.al., (2010) Nature. 466: 835-40, Bartel D.P., (2009) Cell 136; 215, Grimson A et.al., (2007) Mol Cell.27: 91-105.
Webinar 4: Functional Analysisof miRNA
How do miRNAs regulate gene expression?

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How do miRNAs interact with target mRNAs?
Webinar 4: Functional Analysisof miRNA 8
‫‏‬Basis of miRNA–mRNA interaction
Seed region: nucleotides 2–7 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 11–12 continuous base pairs
Result of interaction
 Suppression of gene expression
 Rare cases: increase gene expression
References
 Grimson, A., et al, Mol. Cell 2007, 27, 91-105
 Image From Bartel, D.P., Cell 2009, 136, 215-233
 Guo, H., et al, Nature 2010, 466, 835-840
 Thomson, D.W., et al, Nucleic Acids Res 2011, 1-9

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How to determine miRNA–mRNAinteractions
Webinar 4: Functional Analysisof miRNA 9
Prediction Algorithm Website
TargetScan http://www.targetscan.org/
Pictar http://pictar.mdc-berlin.de/
MicroCosmTargets 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/
‫‏‬Target Prediction is based on:
 Bioinformatics
 Seed region match
 Position in 3′ UTR
 Cross species conservation
 Central sequence homology
 Wet-lab research
 Empirical evidence from microarrays
 Reporter systems
‫‏‬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

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10Webinar 4: Functional Analysisof miRNA
Artificial manipulation of the normal level of a miRNA in a cell

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11Webinar 4: Functional Analysisof miRNA
miRNA mimicand inhibitor experiment workflow

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miScript miRNA Target Protectors4
Agenda
miRNA background1
miScript miRNA Mimics2
miScript miRNA Inhibitors3
12
Controls5

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Webinar 4: Functional Analysisof miRNA 13
miScript miRNA Mimics

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Webinar 4: Functional Analysisof miRNA 14
miScript miRNA Mimic principle

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‫‏‬ Features of miScript miRNA Mimics:
 Double-stranded RNA oligonucleotide
 Functional sequence is the same as the natural mature miRNA
 Transfection results in inhibition comparable to the endogenous miRNA
 Stable in culture for up to 72 hours
Webinar 4: Functional Analysisof miRNA
miScript miRNA Mimics mock endogenous miRNA activity

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Webinar 4: Functional Analysisof miRNA
‫‏‬ Analysis of miScript miRNA Mimic effects:
 Reporter constructs (e.g. luciferase reporters)
 Change in protein expression level
 Change in mRNA expression in some cases
 Downregulation by miRNA does not necessarily lead to degradation of the
target mRNA
Inhibition of endogenous HDAC4 using a miR-1 mimic
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Example of using a miScript miRNAMimic

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Webinar 4: Functional Analysisof miRNA
Target-dependent time course of effect
 Changes in protein expression level
can often be measured after 48–72
hours
 Can depend on different factors
 Time course can differ dependent on
both miRNA and target
 Stability and turnover of the
targets of the same miRNA might
differ
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Analysis of miScript miRNA Mimic effects

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miScript miRNA Target Protectors4
Agenda
miRNA background1
miScript miRNA Mimics2
miScript miRNA Inhibitors3
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Controls5

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Webinar 4: Functional Analysisof miRNA 19
miScript miRNA Inhibitors

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Webinar 4: Functional Analysisof miRNA 20
miScript miRNA Inhibitor principle

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‫‏‬ Features of miScript miRNAInhibitors:
 Single-stranded, chemically modified RNA oligonucleotide
 Designed and modified to ensure efficient inhibition of endogenous miRNA
 Transfection of inhibitor counteracts miRNA-induced silencing
Webinar 4: Functional Analysisof miRNA
miScript miRNA Inhibitors block endogenous miRNA activity

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Webinar 4: Functional Analysisof miRNA
miScript miRNA Inhibitorsare effective after 96 hours
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miScript miRNA Inhibitor stability

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Webinar 4: Functional Analysisof miRNA
55 kD
28 kD Bcl2
TUBB
UT
miR-16
mimic
AllStar
mimic
miR-15
Inhib.
AllStar
Inhib.
miR-16
Inhib.
miR-15
& -16
Inhib.
‫‏‬ Analysis of miScript miRNA Inhibitor effects:
 miRNA targets can be (and often are) regulated by more than one miRNA
 Observing a miRNA inhibitor effect can require inhibiting more than one miRNA
BCL2 is regulated by miR-15a & miR-16
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Example of using a miScript miRNAInhibitor

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Webinar 4: Functional Analysisof miRNA
PDCD4
TUBB
miR-21
mimic
AllStar
mimic
miR-21
Inhib.
AllStar
Inhib.72 kD
55 kD
‫‏‬ Effects of miRNA mimic and inhibitor:
 Transfection of miR-21 mimic leads to further reduction of endogenously
expressed PDCD4
 Transfection of miR-21 mimic leads to an increase of PDCD4 protein
Regulation of PDCD4 by miR-21
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Example of using miScript miRNA mimicsand inhibitors

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miScript miRNA Target Protectors4
Agenda
miRNA background1
miScript miRNA Mimics2
miScript miRNA Inhibitors3
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Controls5

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Webinar 4: Functional Analysisof miRNA 26
miScript miRNA Target Protectors

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Webinar 4: Functional Analysisof miRNA 27
miScript miRNA Target Protector principle

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Webinar 4: Functional Analysisof miRNA
‫‏‬ Features of miScript miRNA Target Protectors:
 Single-stranded, chemically modified RNA oligonucleotide
 Designed and modified to ensure efficient protection of specific mRNAs from
endogenous miRNAs
 Transfection of target protector counteracts miRNA-induced silencing of a
single target
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miScript miRNA Target Protectors protect specific transcripts
Protection of a luc reporter against endogenously expressed miR-16

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Webinar 4: Functional Analysisof miRNA
‫‏‬ Analysis of miScript miRNA Target Protector effects:
 Target protectors are designed against a single target site
 If more than one miRNA targets the site, the target protector counteracts the
effect of both
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Protection of endogenously expressed targets

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Webinar 4: Functional Analysisof miRNA
miR-1 mimic was co-transfected in all samples except UT
‫‏‬ Analysis of miScript miRNA Target Protector effects:
 Target protectors are designed against a single target site
 Other targets affected by the same miRNA are not protected
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Target specificity of miScript miRNA Target Protectors

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Webinar 4: Functional Analysisof miRNA
‫‏‬ Analysis of miScript miRNA Target Protector effects:
 Example: miR-9 causes downregulation of a gene that is important for cell
viability
 After transfection of a miR-9 mimic, an increase of cell death is observed
 miScript Target Protector miR-9 inhibits the downregulation by miR-9, allowing
the cell viability gene to be expressed
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Example of using a miScript miRNATarget Protector

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Target protectors in literature
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Nakashima et al; Journal of Immunology 188, 2012.

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Target protectors in literature
Webinar 4: Functional Analysisof miRNA 33
Nakashima et al; Journal of Immunology 188, 2012.

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Target protectors in literature
Webinar 4: Functional Analysisof miRNA 34
Takeshita et al; International Journal of Oncology 41: 1653-1661. 2012

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Target protectors in literature
Webinar 4: Functional Analysisof miRNA 35
Takeshita et al; International Journal of Oncology 41: 1653-1661. 2012

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miScript miRNA Target Protectors4
Agenda
miRNA background1
miScript miRNA Mimics2
miScript miRNA Inhibitors3
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Controls5

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Mimic
negative control
AllStars Negative Control siRNA
 No homology to any mammalian gene
 Validated for non-effectsin microarray and phenotype assays
 Validated for RISC entry
Mimic
positive control
Syn-hsa-miR-1 mimic
 Not expressed under most culture conditions; only expressed in muscle cells
 To check for optimal conditions by using miScript PCR miR-1
Inhibitor
negative control
miScript Inhibitor Negative Control
 Target the sequence of miScript mimic negative control
Inhibitor
positive control
Anti-hsa-miR-1 inhibitor
 To check for optimal conditions in combination with Syn-hsa-miR-1 mimic
Transfection
control
AllStars Hs Cell Death Control siRNA
 Cell death = successful transfection
 To run in every experiment
Webinar 4: Functional Analysisof miRNA
Controls for miRNA functional studies

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Webinar 4: Functional Analysisof miRNA 38
‫‏‬ Important aspects to consider:
 Are both miRNA and target of interest expressed under the experimental
conditions?
 What detection system is suitable?
 How does the miRNA regulate the target (mRNA or protein level)
 Could the target be regulated by other miRNAs?
 Is the chosen cell line suitable for the expected time frame?
 Does the miRNA affect cell viability?
miRNA functional analysis tips

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Where can I find the QIAGEN products discussed today?
Webinar 4: Functional Analysisof miRNA 39
www.qiagen.com
www.qiagen.com/GeneGlobe

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Webinar 4: Functional Analysisof miRNA 40
QIAGEN Sample to Insight Solutions for miRNA research
Sample Prep
Real-time
PCR Assays
Data
Analysis
Interpretation
 QIAcube
 miRNeasy Mini
 miRNeasy
Micro
 miRNeasy
FFPE
 miRNeasy
Serum / Plasma
 ExoRNeasy
Serum / Plasma
Instruments
 QIAgility
 RotorGene Q
 Compatibility with all
Real-Time PCR instruments
 miScript PCR System
 miScript PreAMP
 miScript Microfluidics
 miScript PCR Arrays
 miScript Primer
Assays
 GeneGlobe
Data Analysis
Center
Kits/
Solutions
 Ingenuity Pathway
Analysis
 miScript Mimics
 miScript Inhibitors
 miScript Target
Protectors

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Webinar 4: Functional Analysisof miRNA 41
Thank you for attending today’s webinar!
Martin Kreutz
Martin.Kreutz@qiagen.com
Contact QIAGEN
1-800-426-8157
BRCsupport@QIAGEN.com
Questions?