reporter assay and q pcr application 2012

Sample & Assay Technologies- 1 -For Internal Use Only

High -Performance Cell-Based Assay and qPCR Technologies for Pathway -Focused Research

Louis Lichten, PhDApplication Scientist

5.10.12

Webinar


Presentation Overview

o QIAGEN’s SABiosciences portfolio

o What is a PCR Array and how do I use it?

o Cignal & Cignal Lenti Pathway Reporters

o Summary


Systems Biology at QIAGEN

DNA

RNA

Protein

Mutation AnalysisChIPMethylationSNP/CNV

Somatic Mutation ArraysEpiTect ChIP-qPCR AssaysMethyl-Profiler qPCR ArraysGenomics Service Core

Discovery Screening

Confirmation

Genomics Service CoreRT2 PCR ArraysmiScript miRNA Arrays & AssaysRT2 qPCR Assays

DetectionKnockdownReporter SystemPhenotypes

Cignal Reporter SystemELISArray KitssiRNA Arrays / shRNA PlasmidsPhosphoprotein Quantitation Kits

Genomics &Regulation

Gene Expression

Gene FunctionProtein Function

Technologies SABiosciences™ ProductsResearch






o Summary


Anatomy of a PCR Array

84 Pathway-Specific Genes of Interest

5 Housekeeping Genes

Genomic DNA Contamination Control

Reverse Transcription Controls (RTC) n=3

Positive PCR Controls (PPC) n=3

96 well version (1 sample/PCR Array)(384 well version available (4 samples/PCR Array)


RT2 Profiler PCR Arrays

.cDNA Synthesis� 45 minutes

.Load Plates (Preferably with 8-Channel Pipettors)

� 2 minutes� Automated Solution = QIAgility

.Run 40 cycle qPCR Program � 2 to 2.5 hours� Compatible with all major

Real Time PCR Instruments

.Upload and Analyze Data� 15 minutes


PCR Arrays for All Biomedical Researchers

Complete PCR Array List

384-Well Custom ArraysDiabetesp53 Signaling

96-Well Custom ArraysAtherosclerosiscAMP / Calcium Signaling

Custom PCR ArraysOther DiseasesMAP Kinase Signaling

Stress & ToxicityInsulin Signaling

Neurogenesis and Neural Stem CellOxidative StressDNA Damage Signaling

Neurotrophins & ReceptorsDrug TransportersJak / Stat Signaling

Neurotransmitter ReceptorsDrug Phase I EnzymesNFkB Signaling

Neuroscience Ion ChannelsDrug MetabolismSignal Transduction PathwayFinder

NeuroscienceToxicology & Drug MetabolismSignal Transduction

ECM & AdhesionToll-like ReceptorsCancer Drug Resistance

Growth FactorsTNF LigandsAngiogenesis

OsteogenesisTh1-Th2-Th3Cancer PathwayFinder

Endothelial Cell BiologyNF-κB Signaling PathwayTumor Metastasis

TGFβ / BMP SignalingChemokines Breast Cancer & Estrogen Receptor

Notch SignalingCommon CytokinesHuman miRNA Array (NEW!)

Wnt SignalingTh17 for Inflammation (NEW!)Cell Cycle

Stem CellsInflammatory CytokinesApoptosis

Development & Stem CellsCytokines & InflammationCancer and Apoptosis

Complete PCR Array List

384-Well Custom ArraysDiabetesp53 Signaling

96-Well Custom ArraysAtherosclerosiscAMP / Calcium Signaling

Custom PCR ArraysOther DiseasesMAP Kinase Signaling

Stress & ToxicityInsulin Signaling

Neurogenesis and Neural Stem CellOxidative StressDNA Damage Signaling

Neurotrophins & ReceptorsDrug TransportersJak / Stat Signaling

Neurotransmitter ReceptorsDrug Phase I EnzymesNFkB Signaling

Neuroscience Ion ChannelsDrug MetabolismSignal Transduction PathwayFinder

NeuroscienceToxicology & Drug MetabolismSignal Transduction

ECM & AdhesionToll-like ReceptorsCancer Drug Resistance

Growth FactorsTNF LigandsAngiogenesis

OsteogenesisTh1-Th2-Th3Cancer PathwayFinder

Endothelial Cell BiologyNF-κB Signaling PathwayTumor Metastasis

TGFβ / BMP SignalingChemokines Breast Cancer & Estrogen Receptor

Notch SignalingCommon CytokinesHuman miRNA Array (NEW!)

Wnt SignalingTh17 for Inflammation (NEW!)Cell Cycle

Stem CellsInflammatory CytokinesApoptosis

Development & Stem CellsCytokines & InflammationCancer and Apoptosis


Accessing PCR Array Content


PCR Array: Functional Gene Groupings


PCR Array Layout


miScript miRNA PCR Arrays

� Human miScript miRNA PCR Array� Mouse miScript miRNA PCR Array� Rat miScript miRNA PCR Array� Dog miScript miRNA PCR Array

� Human, Mouse, Rat:� Brain Cancer � Cancer� Cell Differentiation & Development� Immunopathology � Inflammation� miFinder� Neurological Development & Disease � Serum & Plasma

Complete miRNA genome (miRNome) miScript miRNA Pathway Arrays

SpeciesNumber of

assays(miRBase V16)

Total Number of Plates

96 well format

384 well format

Human 1068 12 3

Mouse 947 12 3

Rat 653 8 2

Dog 277 4 1


Custom PCR Arrays – VERY POPULAR!

Analyze the Genes Most ImportantTo Your Research

• Same reliable and convenient PCR Array product format

• 2 to 3 week turn around time

• Upload and Analyze Data, just like our cataloged arrays


Species Cataloged Array

RNA QC Array

HKG Array Custom Array

Individual Primer Assays

Human Y Y Y Y Y

Mouse Y Y Y Y Y

Rat Y Y Y Y Y

Rhesus macaque No Y Y Y Y

Drosophila No Q2 2012 Q2 2012 Y Y

Dog No Y Y Y H2 2012

Pig No Y Q2 2012 Y H2 2012

Chicken No Q2 2012 Q2 2012 Q2 2012 H2 2012

Cow No Q2 2012 Q2 2012 Q2 2012 H2 2012

Zebrafish No Q2 2012 Q2 2012 Q2 2012 H2 2012

Horse No Q2 2012 Q2 2012 Q2 2012 H2 2012

Arabidopsis No Q2 2012 Q2 2012 Q2 2012 H2 2012

Chinese Hamster No Q2 2012 Q2 2012 Q2 2012 H2 2012

PCR Array Species Expansion


Wet-Bench Validation of ALL qPCR Assays

• SensitivityConstant C(t) value for same amount of template

• Amplification EfficiencyDART-PCR analysis

• Specificity• Single, symmetric dissociation curves• Single band of appropriate size on agarose gel

• Dynamic RangeLinear over 8 logs of template

• NTCNo primer dimer formation


CONCLUSION: Each well on PCR Arrays detects a single gene-specific product.

• Assays on Human TGFβ & BMP Signaling Pathway PCR Array used to analyze BMP genes in Universal Reference RNA.

• Single peak dissociation curves

• Single gel bands of predicted size

• High specificity for genes difficult to design specific primers for.

Exceptional SpecificityExceptional Specificity


Excellent Reproducibility

Reproducibility Among Different Instruments

Human Drug Metabolism PCR Array in Universal Reference RNA

CONCLUSION: The PCR Arrays are SO reproducible that the same raw threshold cycle data can be obtained for the same samples even from different end users at different times or using different instruments.

Reproducibility Among Different Users


Application Example: miRNA Profiling


Summary: PCR Arrays

Complete System for mRNA or miRNA Studies- Four-component system: sample prep, arrays, master mix, first strand

kit, and free data analysis software- Four-hour protocol from RNA to presentation-quality results

Breadth of Pathway Content- More than 100 pathways: human, mouse, rat, monkey, dog- FULLY CUSTOMIZABLE by gene, set, or pathway

VERY RELIABLE Real-Time PCR Performance- Wet-bench validation of EVERY primer assay pre-sale- Melt curve analysis to confirm single amplicon specificity

Hundreds of Peer-Reviewed Articles Using PCR Arrayshttp://www.sabiosciences.com/support_publication.ph p






o Summary


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-1MEF2 MEF2c-myc Myc/Max Nanog NanogNFkB NFkBNotch RBP-JkOct4 Oct4Pax6 Pax6PI3K/AKT FOXOPKC/Ca++ NFATPPAR PPARProgesterone PRRetinoic Acid RARRetinoid X RXRSox2 Sox2SP1 SP1STAT3 STAT3TGF-beta SMAD2/3/4VDRE Vitamin D ReceptorWnt TCF/LEFXenobiotic AhR


Cignal Reporter Assays: Experimental Design


Cignal Finder Arrays


How Does a Cignal Finder Array Work?


J. Biol. Chem. 2006;281: 24085-9.

Application Example: Signal Transduction


Complimentary Arrays: Signal Transduction

•Signal Transduction Pathway Finder

•NFkB Signaling Pathway•NFkB Signaling Targets

•TGFb Signaling Pathway•TGFb Signaling Targets

•WNT Signaling Pathway•WNT Signaling Targets

•Notch Signaling Pathway•Notch Signaling Targets


Cignal Reporters: Benefits & Challenges

Breadth of Pathways and Research Applications- 45 Pathway-Focused Reporter Assays- 4 Application-Specific Cignal Finder 10-Pathway Arrays

Convenience- Ready-to-use, validated, preformatted transient assays

Dual Modalities- Dual-luciferase quantitative endpoint assays- GFP dynamic live cell assays with single cell resolution

Biggest Unmet Need for Cignal ResearchersHow do I use the Cignal Reporters in cells that are difficult or impossible to transfect?


Cignal Pathway Reporter System Products

Easy totransfect cell lines

Primary cells, stem cells,and difficult to transfect cell lines

EndpointAssays

EndpointAssays

Dynamic Live Cell Assays

Dynamic Live Cell Assays

Cignal Dual-Luciferase Reporter Assays

Cignal Lenti Luciferase Reporters

Cignal GFP Reporter Assays

Cignal Lenti GFP Reporters

Single PathwayAssays




10-PathwayArrays

10-PathwayArrays


Cignal Lenti Reporter Assays (35 Pathways)

Pathway Transcription FactorAmino Acid Deprivation ATF4/ATF3/ATF2Androgen Androgen ReceptorAntioxidant Response Nrf2 & Nrf1ATF6 ATF6C/EBP C/EBPcAMP/PKA CREBCell Cycle E2F/DP1DNA Damage p53 EGR1 EGR1ER Stress CBF/NF-Y/YY1Estrogen ER GATA GATA Glucocorticoid 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-1MEF2 MEF2c-myc Myc/MaxNanog NanogNFkB NFkBNotch RBP-JkOct4 Oct4Pax6 Pax6PI3K/AKT FOXOPKC/Ca++ NFATPPAR PPARProgesterone PRRetinoic Acid RARRetinoid X RXRSox2 Sox2SP1 SP1STAT3 STAT3TGFβ SMAD2/3/4VDRE Vitamin D ReceptorWnt TCF/LEFXenobiotic AhR


Cignal Lenti Reporters: Benefits

Transduce any mammalian cell– Primary cells– Stem cells– Difficult-to-transfect cell lines

Transduction-ready– No lentiviral generation– No titering assays

Versatile System – Transient experiments– Generate pathway sensor cell

lines


PASMC (Normal Human Pulmonary Artery Smooth Muscle cells) RAW 264.7 (Mouse leukaemic monocyte macrophage cell line)

C6 (Rat glioma cells) CV 1 (Normal African green monkey kidney fibroblast )

Lenti Reporters: Challenging Cell Types

SRE ReporterRBP-Jk Reporter

NFAT Reporter NFkB Reporter

Ad-Neg Ad-NICD

PMA + Ionomycin LPS

Vehicle

Vehicle Vehicle

PMA + FBS


Lenti Reporters Enable Rapid Pathway Sensor Cell Li ne Generation

NFkB Pathway Sensor Cell Line


Summary: Cignal Lenti Reporters

.Breadth of Pathways and Arrays• 35 Pathway-Focused Reporters• 2 application-focused Cignal Finder Lenti 10-Pathwa y Arrays

.PerformanceExceptional sensitivity, specificity, signal-to-noi se ratio, and reproducibility, using either luciferase or GFP rep orter formats

.High Efficiency Lentiviral Delivery System• Study cell signaling in primary cells, stem cells, and difficult to

transfect cell lines• Rapidly generate pathway sensor cell lines

.Convenience• Ready-to-use, validated reporters• Don’t have to produce or amplify lentivirus in your laboratory


Take Home Message

Tell a better Biological Story from your experiments

• Publish faster and in higher impact journals

• Improve Success Rates of Your Grant Applications

• Expedite Drug Pipeline

• Work SMARTER, NOT Harder


QUESTIONS?

Louis Lichten661.702.3133

[email protected]

Webinar

reporter assay and q pcr application 2012

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