Tpa 2013

Sample & Assay Technologies

SureFIND™ Transcriptome PCR ArrayscDNA Panels to Accelerate Discovery

Samuel J. Rulli, Ph. [email protected]

Discover which miRNA, transcription factor or signaling pathway is regulating your favorite gene’s expression.

Sample & Assay Technologies- 2 -

.Transcriptional Regulation and Mechanisms� What regulates your gene’s expression?� Techniques

.What is a Transcriptome PCR Array?� General Description� Protocol Overview

.Discovery in an Afternoon� Application Example

.Data Analysis and Technical Tips� Easy to use Downloadable Excel Templates

.What’s Next and Summary� Follow-up Experiments

Webinar Topics


GOI

What regulates your gene?

mRNA

DNA

miRNA

TranscriptionalFactors

Upstream Pathway Regulators


How do you identify those regulators?

.Overexpression

.RNAi knockdown

.Chemical Treatments.Promoter Analysis

.Molecular Techniques

.Bioinformatics

mRNA

DNA

×

×


So who knows what regulates your gene-of-interest?


What regulates your gene?

mRNA

DNA

miRNA

TranscriptionalFactors

Upstream Pathway Regulators


miRNA mimic

Approach to Identifying Regulators

miRNA

1) Treat Cells

2) Isolate RNA

3) First Strand cDNA Synthesis

4) qPCR for Gene Expression

1 2 3 4


I can do this once or twice…

Reagent Price Price for 10 / 50 / 90

miRNA mimic $150 - $240 $1500 - $2400 / $7500 - $12000 / $13500- $21600







Webinar Topics


What is a Transcriptome PCR Array?

.Transcriptome PCR Arrays are ready-to-use cDNA panels for discovering the miRNAs, Pathways or Transcription Factors 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 treatment.


Example Plate

cDNA Sample 1

cDNA Sample 2

cDNA Sample 3

cDNA Sample 4

cDNA Sample 5

cDNA Sample 6


What is needed for the Protocol?

.What’s in the box?

.One Transcriptome PCR Array � miRNA mimics � TF knockdown, � Chemical Inhibitors of key Pathway Proteins

.The Researcher Must Supply:

.PCR Thermocycler

Micropipettor

qPCR Master Mix

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

Computer with Microsoft Excel for Data Analysis


Protocol







Webinar Topics


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


Canonical pathway of microRNA (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 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.


How to determine miRNA-mRNA interactionsAlgorithms for predicting miRNA-mRNA interaction

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


How to determine miRNA-mRNA interactionsExperimental techniques

.miRNA Target Screening� Gene expression analysis (inferred targets)

� RNA-seq� Microarrays� qPCR

� Immunoprecipitation (direct targets)� HITS-CLIP� PAR-CLIP� Biotin tagged miRNA

.Gene-Specific Validation� qPCR� Luciferase reporter assays� Western blot� 5’ rapid identification of cDNA ends (5’ RLM-RACE)

.Other techniques� Parallel analysis of RNA ends (modified 5’ RLM-RACE)� Reverse transcription of targets

Image From Chi, S.W., et al, Nature 2009, 13, 479-486


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


Manipulating miRNA function


miScript miRNA Mimics act just like endogenous miRN A

. 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


Example Plate

cDNA Sample 1

cDNA Sample 2

cDNA Sample 3

cDNA Sample 4

cDNA Sample 5

cDNA Sample 6


Transcriptome PCR Array Results for INPPL1






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

Confirm in your biological System







Webinar Topics


Data Analysis


Tips for Success

.Know your Gene� Human Orthologue� Validated qPCR Assay

� Either SYBR Green or Hydrolysis Probe� Single-plex or Multiplex

� Expression Pattern� Is GOI expressed in HeLa, HEK293, MCF…?

.Normalization� Use one or more housekeeping genes� Data Analysis Spreadsheet Simplifies ∆∆Ct calculations







Webinar Topics


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






� ssRNA that disrupt a specific Target-miRNA interaction.Pathway-based Discovery

� Repeat chemical treatment� Dose curve or a Time Course

� Knockdown primary chemical inhibitor target� Contact us -> pair your inhibitor with the correct reagent






� ssRNA that disrupt a specific Target-miRNA interaction.Pathway-based Discovery

� Repeat chemical treatment� Dose curve or a Time Course

� Knockdown primary chemical inhibitor target� Contact us -> pair your inhibitor with the correct reagent

.Transcription Factor-based Discovery � Repeat siRNA treatment

� Different siRNA design� Rescue experiment – Expression vector with target sequence (QIAgenes)


Product Information

Product Contents Cat. No.

SureFIND Transcriptome PCR Array One 96-well PCR plate (A or C format)

336611

Cells Treated With Cell Lines Treated Pathways

miRNA mimetic HeLa CancerImmunopathologyDevelopment & DifferentiationComplete miRNome

Drugs HEK293, HeLa, MCF-7 Chemical Inhibitors

siRNAs MCF-7 Cancer-related Transcription FactorsExpressed Transcription Factors


Where do I find out more?


List of Treatments


Summary

� Largest collection of cDNA panels derived from treated cells

� Choice� One plate packs � Multiple cell lines� Drug treatments, miRNA mimics and siRNAs

� Included Data Analysis Spreadsheet

� Accelerating the Discovery Process


SureFIND Transcriptome PCR Array Pilot Study Offer

Questions?

Contact Technical Support 9 AM – 6 PM Eastern M – FTelephone: 888-503-3187;

Email: [email protected]

Samuel Rulli, Ph.D.: [email protected]

Tpa 2013

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