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Isolation FunctionalizationQuantification
Small Molecules with Big Impact: Functional Studies of miRNA
Martin KreutzScientist, R&[email protected]
Third in a series of QIAGEN webinars on miRNA Spring 2011
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Isolation FunctionalizationQuantification
QIAGEN miRNA Webinar Series1. The miRNA Revolution2. miRNA Expression Analysis3. Functional Studies of miRNA
Previous webinars can be found at www.QIAGEN.com
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Why Study miRNA?
Exponential growth in miRNA Studies over ten year period
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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Functional Studies of miRNAs
Introduction1
miScript miRNA Mimics2
miScript miRNA Inhibitors3
miScript miRNA Target Protectors4
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microRNA Biogenesis: Canonical Pathway
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CYTOPLASM
mRNA cleavage
NUCLEUSmicroRNA Gene
POL II
mature miRNA
Pre-miRNA
RISC Assembly
Drosha-DGCR8
Exportin
Exportin
DICER-TRBP
Pri-miRNA
Ago
RISC
DNA
Translational RepressionmRNA degradation
High homology Partial homology
� 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 imperfect base pairing to thetarget mRNA, lead to translational repression and/or mRNA degradation
Krol, J et.al., (2010) Nature Rev Genetics, 11, 597; Winter, J. et.al., (2009) Nature Cell Biology, 11, 228 Borchert, G.M., et.al., (2006)
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Stronger
Weaker
. Target Prediction is based on:� Seed region match
� Position in 3’ UTR
� Cross species conservation� Central sequence homology
� Empirical evidence from microarrays
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.
What Genes Do miRNAs RegulatePrediction of miRNA-mRNA Interaction
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How Do miRNAs Regulate Gene ExpressionmiRNA - mRNA Interactions
Mechanisms of Gene Silencing by miRNA� Ribosome drop-off� Deadenylation and degradation� miRNA sequestration
Translational Repressionand/or
Transcript DegradationP Body Sequestration
Translational Repressionand/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.
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microRNA Functional StudiesHow does it work?
Artificial manipulation of the normal level of a miRNA in a cell
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Experimental WorkflowmiRNA mimics, inhibitors and target protectors
miRNA Mimic, Inhibitor or
Target Protector
Luciferase assay Real time PCR Western blot
Incubation 37 °C, 5% CO2
24h (up to 96h)
Incubation 37 °C, 5% CO2
24h up to 96h or more
Luciferase reporterconstruct
Attractene HiPerFectCotransfection Transfection or
Cotransfection
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Functional Studies of miRNAs
Introduction1
miScript miRNA Mimics2
miScript miRNA Inhibitors3
miScript miRNA Target Protectors4
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miScript mimics can regulate endogenous targets of a given miRNAmiScript mimics can regulate endogenous targets of a given miRNA
miScript miRNA mimicsPrinciple
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miScript miRNA mimicsInhibition of luciferase reporter using a miR-1 mim ic
Reliable, reproducible target regulation after transfectionof miScript miR-1 mimic
Reliable, reproducible target regulation after transfectionof miScript miR-1 mimic
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miScript miRNA mimicsInhibition of Endogenous HDAC4 using a miR-1 Mimic
.No change observed in. HDAC4 mRNA level
.No change observed in. HDAC4 mRNA level
Downregulation of HDAC4 protein using miR-1 mimic
Downregulation of HDAC4 protein using miR-1 mimic
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miScript miRNA mimicTarget-Dependant Time Course of Effect
Targets of miR-1 are regulated differently after transfection of a miR-1 mimicTargets of miR-1 are regulated differently after transfection of a miR-1 mimic
Post transfection
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130
55
55
55
Mock pos neg pos neg pos neg pos neg
24h 48h 72h 96h
HDAC 4
PDCD 4
tubulin
tubulin
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Functional Studies of miRNAs
Introduction1
miScript miRNA Mimics2
miScript miRNA Inhibitors3
miScript miRNA Target Protectors4
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miScript miRNA inhibitorPrinciple of Action
miScript miRNA inhibitors block the regulation of all targets of a given miRNAmiScript miRNA inhibitors block the regulation of all targets of a given miRNA
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miRNA InhibitionQIAGEN miRNA inhibitors effective after 96 hours
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miRNA InhibitionBCL2 is regulated by miR-15a & miR-16
55 kD
28 kD Bcl2
TUBB
UTmiR-16mimic
AllStarmimic
miR-15Inhib.
AllStarInhib.
miR-16Inhib.
miR-15& -16 Inhib.
Co-transfection of miRNA inhibitors against miR-15a and miR-16 is requiredto successfully inhibit regulation of BCL2
Co-transfection of miRNA inhibitors against miR-15a and miR-16 is requiredto successfully inhibit regulation of BCL2
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miRNA InhibitionRegulation of PDCD4 by miR-21
PDCD4
TUBB
miR-21mimic
AllStarmimic
miR-21Inhib.
AllStarInhib.72 kD
55 kD
Transfection of miR-21 mimic leads to further reduction of endogenouslyexpressed PDCD4
Transfection of miR-21 mimic leads to an increase of PDCD4 protein
Transfection of miR-21 mimic leads to further reduction of endogenouslyexpressed PDCD4
Transfection of miR-21 mimic leads to an increase of PDCD4 protein
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Functional Studies of miRNAs
Introduction1
miScript miRNA Mimics2
miScript miRNA Inhibitors3
miScript miRNA Target Protectors4
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miScript Target ProtectorPrinciple of Action
miScript Target Protector enables confirmation of a specific miRNA target miScript Target Protector enables confirmation of a specific miRNA target
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miScript Target Protector Protection of a luc reporter against endogenously ex pressed miR-16
Reliable, reproducible miRNA inhibition after transfectionof miScript Target Protector miR-16
Reliable, reproducible miRNA inhibition after transfectionof miScript Target Protector miR-16
RLU
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miScript Target ProtectorProtection of endogenously expressed targets
Protection of endogenously expressed Bcl2 after transfectionof miScript Target Protector miR-15/16 Bcl2
Protection of endogenously expressed Bcl2 after transfectionof miScript Target Protector miR-15/16 Bcl2
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miScript Target ProtectorProtection Against Phenotypic Effects
Determination of miRNA role in a pathway or process
Example : miR-9 causes down regulation of a gene that is important for cell viability
miScript Target Protector miR-9 inhibits the down regulation by miR-9allowing the cell viability gene to be expressed
miScript Target Protector miR-9 inhibits the down regulation by miR-9allowing the cell viability gene to be expressed
Untreated cells Cell death caused by miR-9
Cell survival after treatment with TP
TP = Target Protector
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miScript Target ProtectorTarget specificity
55 kD
130 kD
ADAR
TUBB
HDAC4
TUBB
UT 1
Negctrl
0.10.5 0.05
ADAR protector [µM]UT 1
Negctrl
0.10.5 0.05
ADAR protector [µM]
miR-1 mimic was co-transfected in all samples except UT
Specific miRNA inhibition after transfectionof miScript Target Protector miR-1 - ADARSpecific miRNA inhibition after transfectionof miScript Target Protector miR-1 - ADAR
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miScript miRNA Mimic & Inhibitor Controls
Anti-hsa-miR-1 inhibitor � To check for optimal conditions in combination with Syn-hsa-miR-1 mimic
� miScript Target Protector Negative Control� miScript Target Protector Positive Control
Inhibitor positive control
Target Protector
AllStars Hs Cell Death Control siRNA� Cell death = successful transfection� To run in every experiment
Transfection control
miScript Inhibitor Negative Control� Target the sequence of miScript mimic negative control
Inhibitor negative control
Syn-hsa-miR-1 mimic� Not expressed under most cell culture conditions, only expressed in muscle cells � To check for optimal conditions by using miScript PCR miR-1
Mimic positive 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 negative control
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www.qiagen.com/GeneGlobe
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A Complete Solution for miRNA Research
A complete offering for miRNA research!A complete offering for miRNA research!
Rotor-Gene QQIAgility
PAXgene Tissue miRNA Kit
PAXgene Blood miRNA Kit
QIAamp Circulating Nucleic Acid Kit
miRNeasy Protect Animal Blood Kit
miScript Pre-miRNA Assays
QIAcube
miScript PCR Controls
SABiosciences RT 2 miRNA PCR Arrays RT2 SYBR Green Mastermixes
miScript Target Protectors
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Thank you!
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