Sample & Assay Technologies SureFIND™ Transcriptome PCR Arrays cDNA Panels to Accelerate Discovery Samuel J. Rulli, Ph. D. [email protected]Discover which miRNA, transcription factor or signaling pathway is regulating your favorite gene’s expression.
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Sample & Assay Technologies
SureFIND™ Transcriptome PCR ArrayscDNA Panels to Accelerate Discovery
.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
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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.
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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
.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
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Protocol
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.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
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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
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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 interactionsAlgorithms for predicting miRNA-mRNA interaction
.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
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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 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
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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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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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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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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
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.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
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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…?
.Normalization� Use one or more housekeeping genes� Data Analysis Spreadsheet Simplifies ∆∆Ct calculations
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.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
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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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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.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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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.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