mi rna functional analysis 2013

Sample & Assay Technologies

Functional analysis of miRNA

Samuel J. Rulli, Jr. [email protected]

Sample & Assay Technologies- 2 -

Topics for webinar

What is microRNA?Manipulation of miRNA in cell lines using reporter assays

miRNA Mimics InhibitorsTarget protectors

Screening your target gene for miRNA regulationReady-to-use cDNA panels

Identification of miRNA regulation by qPCRmiRNA Targets PCR Arrays

SummaryQ & A session


What Is Micro RNA?

Endogenously expressed small functional RNAs (~ 21nt -24 nt)

Regulate mRNA expression post-transcriptionally Involves the formation of RISC (RNA induced Silencing Complex) Translational Repression and or mRNA degradation

Only partial sequence complementarity required A given miRNA can regulate multiple mRNAs A given mRNA can be regulated by multiple miRNAs Another layer of complexity for regulating gene expression

Aberrant expression of miRNAs is associated with many diseasese.g. Many different Cancers

Ω

Seed region

5’ 3’

miRNA

Target mRNA

5’3’CDS

RISC

Ω

Seed region

5’ 3’

miRNA

Target mRNA

5’3’CDS

RISC

Ω

Seed region

5’ 3’

miRNA

Target mRNA

5’3’CDS

RISC


Canonical pathway of microRNA 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 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 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


A growing picture of miRNA dysregulation in cancer, developmental and metabolic pathways

.In the last 5 years, a substantial number of studies and reviews have associated

.the presence of various miRNAs with cell proliferation, resistance to apoptosis, and

.differentiation in cancer cells.

.For example:

Deletions of miRNA-regulated genes have been detected in more than 65% of chronic lymphocytic leukemia cases, in 50% of mantle-cell lymphomas, in 16% to 40% of multiple myelomas, and in 60% of prostate cancers.

Other miRNA abnormalities have been reported in a wide variety of Human neoplasms, including other hematologic malignancies such as

promyelocytic leukemia Benign tumors such as leiomyoma and pituitary adenoma Multiple types of carcinomas, including pancreatic, esophageal,

thyroid, lung, and breast Neuroblastomas and glioblastomas


Topics for webinar







Manipulation of miRNA in cell lines

Functional interactions between miRNA and mRNA can be follow using:

Reporter assays: reporter gene has miRNA site in 3’ UTR which decreases reporter gene translation or causes transcriptional degradation

Real-time PCR: monitors transcriptional degradation of mRNA

Western blot: monitors transcriptional or translational changes

Pathway reporter assay: monitors the pathway activity of specific transcriptionFactors.


miRNA Mimics & Inhibitors: How do they work?

Transfection of Mimics

Transfection of Inhibitors

RISC

Target Expression Level





RISC


Mimics increase miRNA activity


Comparable down-regulation by mimics and endogenous miRNAs

Hela cells with reporter assays for miR-1 or miR-16. miR-16 is endogenously Expressed, while miR-1 was introduced using a miRNA mimic


Stability of miRNA mimics

miRNA mimics are stable for at least 72 hours.


HDAC4

HDAC4 is a target of mir-1

HDAC4 had 2 miR-1 interaction sites. HeLa cells transfected with mir-1 show downRegulation of HDAC4 at the mRNA and protein levels.





RISC


Inhibitor decrease miRNA activity


mir-16 is decreased by miR-16 inhibitor in HeLa cells by reporter assay


Application Example: miRNA & Pathway Activity

.The let-7 family of miRNAs is widely viewed as playing a role in tumor suppression.

.Expression of let-7 family members is downregulated in many cancer types and during tumor progression in comparison to normal tissue.

.Humans have 10 mature let-7 family sequences (let-7a, 7b, 7c, 7d, 7e, 7f, 7g, 7i, miR-98, and miR-202)

.Interestingly, certain let-7 species are also known to be upregulated in some cancer types. These data on the expression of let-7 in various cancers indicate that individual let-7 family members can have different activities.

.Question: Which let-7 family members effect cell cy cle progression and how?


Cignal Reporters: What You Need

• Cignal Cell Cycle Pathway ReporterMeasures E2F/DP1 Activity

• Transfection of HEK293 cells

• Biological Test •Let-7 family mimics

• Dual-Luciferase Assay


Cignal Reporter Assays (45 Pathways)

Pathway Transcription FactorAmino Acid Deprivation ATF4/ATF3/ATF2Androgen Androgen ReceptorAntioxidant Response Nrf2 & Nrf1ATF6 ATF6C/EBP C/EBPcAMP/PKA CREBCell Cycle E2F/DP1DNA Damage p53EGR1 EGR1ER Stress CBF/NF-Y/YY1Estrogen ERGATA GATAGlucocorticoid GRHeat Shock Response HSFHeavy Metal Stress MTF1Hedgehog GLIHNF4 HNF4Hypoxia HIF-1Interferon Regulation IRF-1Type I Interferon STAT1/STAT2Interferon Gamma STAT1/STAT1KLF4 KLF4Liver X LXR

Pathway Transcription FactorMAPK/ERK Elk-1/SRFMAPK/JNK AP-1 MEF2 MEF2c-myc Myc/MaxNanog NanogNFκB NFκBNotch RBP-Jk Oct4 Oct4Pax6 Pax6PI3K/AKT FOXOPKC/Ca++ NFATPPAR PPARProgesterone PRRetinoic Acid RARRetinoid X RXRSox2 Sox2SP1 SP1STAT3 STAT3TGFβ SMAD2/3/4 Vitamin D Vitamin D ReceptorWnt TCF/LEFXenobiotic AhR


Let-7 Family Members and the Cell Cycle


Topics for webinar







miRNA mimic

Approach to Identifying miRNA Regulators

miRNA

1) Treat Cells

2) Isolate RNA

3) First Strand cDNA Synthesis

4) qPCR for Gene Expression

1 2 3 4


Transcriptome cDNA panels

.Transcriptome PCR Arrays are ready-to-use cDNA panels for discovering the miRNAs, that regulate the expression of any gene.

.Each Transcriptome PCR Array is a panel of Transcriptomes or cDNAs: 90 Experimental cDNA samples 4 Vehicle Control cDNA samples 2 PCR Controls (only for SYBR green reactions)

.Each experimental cDNA sample is derived from cells treated with a different miRNA mimic treatment.


Example Transcriptome PCR Array

cDNA Sample 1

cDNA Sample 2

cDNA Sample 3

cDNA Sample 4

cDNA Sample 5

cDNA Sample 6


Materials needed for miRNA discovery

.Transcriptome PCR Array (1 for each GOI or HKG)

.The Research Also Needs:

.qPCR Thermocycler

Micropipettor

qPCR Master Mix

qPCR Assay (either SYBR® green or Probe-based Assays)

Computer with Microsoft Excel for Data Analysis


SureFIND Protocol


miRNA pathways available

• Cancer miRNAs

• Immunopathology

• Development and differentiation

• Complete miRNome (~900 miRNAs)


Application Example: miRNA regulation of INPPL1

.INPPL1, also known as SHIP2, is a SH2-containing 5'-inositol phosphatase

.Converts PIP3 to PtdIns(3,4)P2, which negatively regulates PI3K signaling

.Linked to Carcinogenesis


Site Predictions for INPPL1

TargetScan PicTar miRANDA

miR-10 miR-184 miR-18a

miR-135 miR-205 miR-18b

miR-146 miR-342-3p

miR-184 miR-642

miR-18ab miR-671-3p

miR-205

miR-27ab

miR-29abc


Cancer miRNA Transcriptome PCR Array Results for INPPL1


Site Predictions for INPPL1

TargetScan PicTar miRANDA

miR-10 miR-184 miR-18a

miR-135 miR-205 miR-18b

miR-146 miR-342-3p

miR-184 miR-642

miR-18ab miR-671-3p

miR-205

miR-27ab

miR-29abc


Compare Results with Research Community

What’s really exciting….

Three new miRNAs that play a role in INPPL1 express ion

mir-193bmir-138mir-373


Now what?............. Suggestions to Validate your “Hits”

.miRNA-based Discovery Perform a miScript miRNA mimic experiment

Identical Gain-Of-Function experiment Perform a miScript miRNA inhibitor experiment

Loss-of-Function Experiment Peform a miScript Target Protector experiment

ssRNA that disrupt a specific Target-miRNA interaction


Topics for webinar







Identification of transcriptional degradation using qPCR: RT2 Profiler microRNA Targets PCR Arrays



RISC



• 84 Experimentally verified or predicted targets of miRNA

• 5 Housekeeping Genes

• Genomic DNA Contamination Control

• Reverse Transcription Controls (RTC) n=3

• Positive PCR Controls (PPC) n=3

Supported PCR Array Formats• 96-Well Plate• 384-Well Plate• 100-Well Disc• 96x96 Chip

miR-21 Targets PCR Array

Anatomy of a RT2 Profiler microRNA Targets PCR Arrays


Human Mouse RatmiR-21 miR-21 miR-21

miR-155 miR-155

miR-17 & miR-20a miR-17 & miR-20a miR-17 & miR-20a

miR-146a miR-146a miR-146a

miR-1 & miR-206 miR-1a & miR-206 miR-1 & miR-206

miR-34a miR-34a miR-34a

miR-221 & miR-222 miR-221 & miR-222 miR-221 & miR-222

let-7 let-7 let-7

Available RT² Profiler microRNA Targets PCR Arrays:

Custom miRNA targets arrays are possible based on your miRNA of interest


How RT2 Profiler PCR Arrays Work

. cDNA Synthesis (C-03 kit)– Genomic DNA Removal Step (5 min.)– Reverse Transcription Step (20 min.)

. Load Plates 1 Sample per PCR Array 2 minutes with multi-channel pipet

. Run 40 cycle qPCR Program Standard cycling conditions for all

Real Time PCR Instruments 2 hours

. Upload and Analyze Data 15 minutes from Raw Ct to Fold Change Data


Data Analysis

. Web-Based Software No installation or licensing needed

. From Raw Ct Values to Fold Change Results ∆∆C(t) Method

. Multiple Analysis Formats Scatter Plot Volcano Plot Multi-Group Plot Clustergram

Also available as downloadable Excel templates

Scatter Plot

Volcano Plot

Clustergram


Summary

Global miRNA function can by manipulated by using mimics and inhibitors

Specific miRNA/mRNA interactions can be studied using target protectors

Pathway or transcription factor assays can be used to follow miRNA affects onOn cell biology

RT2 miRNA Targets PCR Arrays facilitate screening miRNA target genes using verified qPCR Assays


Questions, Comments, Concern or Suggestions?

Answers to your technical and biological questions:

Technical Support: 888-503-3187 [email protected]

Samuel [email protected]

Copy of slides? Type slides into questions box or fill out survey on exitWant someone to call you? Type contact me into questions boxWant to try something? Request demos in the questions box.

miRNA mimics, inhibitors or target protectorsCignal reporter assayscDNA panelsmiRNA Targets RT2 Profiler PCR Arrays

mi rna functional analysis 2013

Documents

manipulation of mirna

given mirna

sample assay technologies

mirna site

deletions of mirna

mirna abnormalities

mirna regulation ready

mirna pathway activity