small molecules with big impact

Sample & Assay Technologies- 1 -

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


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


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


microRNA Biogenesis: Canonical Pathway

- 2 -

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)


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


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.


microRNA Functional StudiesHow does it work?

Artificial manipulation of the normal level of a miRNA in a cell


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



Introduction1





miScript mimics can regulate endogenous targets of a given miRNAmiScript mimics can regulate endogenous targets of a given miRNA

miScript miRNA mimicsPrinciple


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


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


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

250

130

55

55

55

Mock pos neg pos neg pos neg pos neg

24h 48h 72h 96h

HDAC 4

PDCD 4

tubulin

tubulin



Introduction1





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


miRNA InhibitionQIAGEN miRNA inhibitors effective after 96 hours


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


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



Introduction1





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


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


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


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


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


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


www.qiagen.com/GeneGlobe


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


Thank you!

small molecules with big impact

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