Tpa 2013

SureFIND™ Transcriptome PCR Arrays
cDNA Panels to Accelerate Discovery
Discover which miRNA, transcription factor or signaling pathway is
regulating your favorite gene’s expression.

Samuel J. Rulli, Ph. D.
Samuel.Rulli@QIAGEN.com
Sample & Assay Technologies

Webinar Topics

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

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What is a Transcriptome PCR Array?
General Description
Protocol Overview

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Discovery in an Afternoon
Application Example

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Data Analysis and Technical Tips
Easy to use Downloadable Excel Templates

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What’s Next and Summary
Follow-up Experiments

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What regulates your gene?

miRNA

Upstream Pathway
Regulators

mRNA

GOI

DNA
Transcriptional
Factors
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How do you identify those regulators?

Bioinformatics

Overexpression

.

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Promoter Analysis

Chemical Treatments

.

.

×

mRNA

RNAi knockdown

Molecular
Techniques

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×

DNA

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

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What regulates your gene?

miRNA

Upstream Pathway
Regulators

mRNA

DNA
Transcriptional
Factors
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Approach to Identifying Regulators

miRNA

1

miRNA mimic

2

3

4

1) Treat Cells
2) Isolate RNA
3) First Strand cDNA Synthesis
4) qPCR for Gene Expression

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I can do this once or twice…

Reagent
miRNA mimic

Price
$150 - $240

Price for 10 / 50 / 90
$1500 - $2400 / $7500 - $12000 / $13500- $21600

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Webinar Topics

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Techniques

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General Description
Protocol Overview

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Application Example

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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)

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

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Example Plate

cDNA Sample 1
cDNA Sample 2
cDNA Sample 3
cDNA Sample 4
cDNA Sample 5
cDNA Sample 6

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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

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The Researcher Must Supply:

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PCR Thermocycler

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Micropipettor
qPCR Master Mix
qPCR Assay (either SYBR® green or Probe-based Assays)
Computer with Microsoft Excel for Data Analysis

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Protocol

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Webinar Topics

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Techniques

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General Description
Protocol Overview

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Application Example

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Application Example: miRNA regulation of INPPL1
INPPL1, also known as SHIP2, is a SH2-containing 5'-inositol phosphatase

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

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Linked to Carcinogenesis

.

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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.

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How to determine miRNA-mRNA interactions
Algorithms for predicting miRNA-mRNA interaction

Target Prediction is based on:
Bioinformatics

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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

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Seed region match
Position in 3’ UTR
Cross species conservation
Central sequence homology

Wet lab research
Empirical evidence from microarrays
Reporter systems
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/
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How to determine miRNA-mRNA interactions
Experimental techniques

miRNA Target Screening

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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

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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
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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

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Manipulating miRNA function

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miScript miRNA Mimics act just like endogenous miRNA

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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

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Example Plate

cDNA Sample 1
cDNA Sample 2
cDNA Sample 3
cDNA Sample 4
cDNA Sample 5
cDNA Sample 6

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Transcriptome PCR Array Results for INPPL1

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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

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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

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Compare Results with Research Community

What’s really exciting….
Three new miRNAs that play a role in INPPL1 expression
mir-193b
mir-138
mir-373

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Confirm in your
biological System


Webinar Topics

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Techniques

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General Description
Protocol Overview

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Application Example

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Data Analysis

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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…?

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

.

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Webinar Topics

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Techniques

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General Description
Protocol Overview

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Application Example

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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

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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

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.

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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)

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.

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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

Cancer
Immunopathology
Development & Differentiation
Complete miRNome

Drugs

HEK293, HeLa, MCF-7

Chemical Inhibitors

siRNAs

MCF-7

Cancer-related Transcription Factors
Expressed Transcription Factors

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Where do I find out more?

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List of Treatments

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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

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SureFIND Transcriptome PCR Array Pilot Study Offer

Questions?
Contact Technical Support 9 AM – 6 PM Eastern M – F
Telephone: 888-503-3187;
Email: support@SABiosciences.com

Samuel Rulli, Ph.D.: Samuel.Rulli@qiagen.com

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