Functional Analysis of miRNA: miRNA and its Role in Human Disease Webinar Series Part 4
-
Upload
qiagen -
Category
Healthcare
-
view
809 -
download
2
Transcript of Functional Analysis of miRNA: miRNA and its Role in Human Disease Webinar Series Part 4
Sample to Insight
Functional analysis of miRNA
Martin Kreutz
Scientist, Product Development
Sample to Insight
Legal disclaimer
Webinar 4: Functional Analysis of 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.
Sample to Insight
Welcome to our four-part webinar series on miRNAs
Webinar 4: Functional Analysis of 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
Sample to Insight
miScript miRNA Target Protectors4
Agenda
miRNA background1
miScript miRNA Mimics2
miScript miRNA Inhibitors3
4
Controls5
Sample to Insight
5
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 Analysis of miRNA
Why assess the function of microRNAs (miRNAs)?
Sample to Insight
6Webinar 4: Functional Analysis of miRNA
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
7
Mechanisms of gene silencing by miRNA:
Ribosome drop-off
Deadenylation and degradation
miRNA sequestration
Translational Repression
and/or
Transcript DegradationP Body Sequestration
Translational Repression
and/or
Transcript DegradationP 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 Analysis of miRNA
How do miRNAs regulate gene expression?
Sample to Insight
How do miRNAs interact with target mRNAs?
Webinar 4: Functional Analysis of 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
end also contributes (extensive pairing is rare)3′
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
Sample to Insight
How to determine miRNA–mRNA interactions
Webinar 4: Functional Analysis of miRNA 9
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/
: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
Sample to Insight
10Webinar 4: Functional Analysis of miRNA
Artificial manipulation of the normal level of a miRNA in a cell
Sample to Insight
11Webinar 4: Functional Analysis of miRNA
miRNA mimic and inhibitor experiment workflow
Sample to Insight
miScript miRNA Target Protectors4
Agenda
miRNA background1
miScript miRNA Mimics2
miScript miRNA Inhibitors3
12
Controls5
Sample to Insight
15
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 Analysis of miRNA
miScript miRNA Mimics mock endogenous miRNA activity
Sample to Insight
Webinar 4: Functional Analysis of 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
16
Example of using a miScript miRNAMimic
Sample to Insight
Webinar 4: Functional Analysis of 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
17
Analysis of miScript miRNA Mimic effects
Sample to Insight
miScript miRNA Target Protectors4
Agenda
miRNA background1
miScript miRNA Mimics2
miScript miRNA Inhibitors3
18
Controls5
Sample to Insight
21
Features of miScript miRNA Inhibitors:
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 Analysis of miRNA
miScript miRNA Inhibitors block endogenous miRNA activity
Sample to Insight
Webinar 4: Functional Analysis of miRNA
miScript miRNA Inhibitors are effective after 96 hours
22
miScript miRNA Inhibitor stability
Sample to Insight
Webinar 4: Functional Analysis of miRNA
55 kD
28 kD Bcl2
TUBB
UTmiR-16
mimicAllStar
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
23
Example of using a miScript miRNAInhibitor
Sample to Insight
Webinar 4: Functional Analysis of 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
24
Example of using miScript miRNA mimics and inhibitors
Sample to Insight
miScript miRNA Target Protectors4
Agenda
miRNA background1
miScript miRNA Mimics2
miScript miRNA Inhibitors3
25
Controls5
Sample to Insight
Webinar 4: Functional Analysis of miRNA 27
miScript miRNA Target Protector principle
Sample to Insight
Webinar 4: Functional Analysis of 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
28
miScript miRNA Target Protectors protect specific transcripts
Protection of a luc reporter against endogenously expressed miR-16
Sample to Insight
Webinar 4: Functional Analysis of 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
29
Protection of endogenously expressed targets
Sample to Insight
Webinar 4: Functional Analysis of 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
30
Target specificity of miScript miRNA Target Protectors
Sample to Insight
Webinar 4: Functional Analysis of 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
31
Example of using a miScript miRNATarget Protector
Sample to Insight
Target protectors in literature
Webinar 4: Functional Analysis of miRNA 32
Nakashima et al; Journal of Immunology 188, 2012.
Sample to Insight
Target protectors in literature
Webinar 4: Functional Analysis of miRNA 33
Nakashima et al; Journal of Immunology 188, 2012.
Sample to Insight
Target protectors in literature
Webinar 4: Functional Analysis of miRNA 34
Takeshita et al; International Journal of Oncology 41: 1653-1661. 2012
Sample to Insight
Target protectors in literature
Webinar 4: Functional Analysis of miRNA 35
Takeshita et al; International Journal of Oncology 41: 1653-1661. 2012
Sample to Insight
miScript miRNA Target Protectors4
Agenda
miRNA background1
miScript miRNA Mimics2
miScript miRNA Inhibitors3
36
Controls5
Sample to Insight
37
Mimic
negative control
AllStars Negative Control siRNA
No homology to any mammalian gene
Validated for non-effects in 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 Analysis of miRNA
Controls for miRNA functional studies
Sample to Insight
Webinar 4: Functional Analysis of 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
Sample to Insight
Where can I find the QIAGEN products discussed today?
Webinar 4: Functional Analysis of miRNA 39
www.qiagen.com
www.qiagen.com/GeneGlobe
Sample to Insight
Webinar 4: Functional Analysis of miRNA 40
QIAGEN Sample to Insight Solutions for miRNA research
Sample PrepReal-time
PCR Assays
Data
AnalysisInterpretation
QIAcube
miRNeasy Mini
miRNeasy Micro
miRNeasy FFPE
miRNeasySerum / Plasma
ExoRNeasySerum / Plasma
Instruments
QIAgilityRotorGene QCompatibility 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 MimicsmiScript InhibitorsmiScript Target Protectors
Sample to Insight
Webinar 4: Functional Analysis of miRNA 41
Thank you for attending today’s webinar!
Martin Kreutz
Contact QIAGEN
1-800-426-8157
Questions?