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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
Sample & Assay Technologies- 2 -For Internal Use Only
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
Sample & Assay Technologies- 3 -For Internal Use Only
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
Sample & Assay Technologies- 4 -For Internal Use Only
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
Sample & Assay Technologies- 5 -For Internal Use Only
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)
Sample & Assay Technologies- 6 -For Internal Use Only
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
Sample & Assay Technologies- 7 -For Internal Use Only
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
Sample & Assay Technologies- 8 -For Internal Use Only
Accessing PCR Array Content
Sample & Assay Technologies- 9 -For Internal Use Only
PCR Array: Functional Gene Groupings
Sample & Assay Technologies- 10 -For Internal Use Only
PCR Array Layout
Sample & Assay Technologies- 11 -For Internal Use Only
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
Sample & Assay Technologies- 12 -For Internal Use Only
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
Sample & Assay Technologies- 13 -For Internal Use Only
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
Sample & Assay Technologies- 14 -For Internal Use Only
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
Sample & Assay Technologies- 15 -For Internal Use Only
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
Sample & Assay Technologies- 16 -For Internal Use Only
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
Sample & Assay Technologies- 17 -For Internal Use Only
Application Example: miRNA Profiling
Sample & Assay Technologies- 18 -For Internal Use Only
Application Example: miRNA Profiling
Sample & Assay Technologies- 19 -For Internal Use Only
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
Sample & Assay Technologies- 20 -For Internal Use Only
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
Sample & Assay Technologies- 21 -For Internal Use Only
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
Sample & Assay Technologies- 22 -For Internal Use Only
Cignal Reporter Assays: Experimental Design
Sample & Assay Technologies- 23 -For Internal Use Only
Cignal Finder Arrays
Sample & Assay Technologies- 24 -For Internal Use Only
How Does a Cignal Finder Array Work?
Sample & Assay Technologies- 25 -For Internal Use Only
J. Biol. Chem. 2006;281: 24085-9.
Application Example: Signal Transduction
Sample & Assay Technologies- 26 -For Internal Use Only
Application Example: Signal Transduction
Sample & Assay Technologies- 27 -For Internal Use Only
Application Example: Signal Transduction
Sample & Assay Technologies- 28 -For Internal Use Only
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
Sample & Assay Technologies- 29 -For Internal Use Only
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?
Sample & Assay Technologies- 30 -For Internal Use Only
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
Single PathwayAssays
Single PathwayAssays
Single PathwayAssays
10-PathwayArrays
10-PathwayArrays
Sample & Assay Technologies- 31 -For Internal Use Only
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
Sample & Assay Technologies- 32 -For Internal Use Only
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
Sample & Assay Technologies- 33 -For Internal Use Only
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
Sample & Assay Technologies- 34 -For Internal Use Only
Lenti Reporters Enable Rapid Pathway Sensor Cell Li ne Generation
NFkB Pathway Sensor Cell Line
Sample & Assay Technologies- 35 -For Internal Use Only
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
Sample & Assay Technologies- 36 -For Internal Use Only
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
Sample & Assay Technologies- 37 -For Internal Use Only
QUESTIONS?
Louis Lichten661.702.3133
Webinar