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Semelhante a Reporter assay and q pcr application 2012
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Reporter assay and q pcr application 2012
- 1. Webinar High-Performance Cell-Based Assay and qPCR Technologies for Pathway-Focused Research Louis Lichten, PhD Application Scientist 5.10.12 For Internal Use Only -1- Sample & Assay Technologies
- 2. 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 For Internal Use Only -2- Sample & Assay Technologies
- 3. Systems Biology at QIAGEN Research DNA RNA Gene Expression Protein Gene Function Protein Function For Internal Use Only SABiosciences™ Products Somatic Mutation Arrays EpiTect ChIP-qPCR Assays Methyl-Profiler qPCR Arrays Genomics Service Core Discovery Screening Confirmation Genomics & Regulation Technologies Mutation Analysis ChIP Methylation SNP/CNV Genomics Service Core RT2 PCR Arrays miScript miRNA Arrays & Assays RT2 qPCR Assays Detection Knockdown Reporter System Phenotypes Cignal Reporter System ELISArray Kits siRNA Arrays / shRNA Plasmids Phosphoprotein Quantitation Kits -3- Sample & Assay Technologies
- 4. 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 For Internal Use Only -4- Sample & Assay Technologies
- 5. 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) For Internal Use Only -5- Sample & Assay Technologies
- 6. RT2 Profiler PCR Arrays cDNA Synthesis 45 minutes . Load Plates (Preferably with 8Channel 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 . For Internal Use Only -6- Sample & Assay Technologies
- 7. PCR Arrays for All Biomedical Researchers Cancer and Apoptosis Cytokines & Inflammation Development & Stem Cells Apoptosis Inflammatory Cytokines Stem Cells Cell Cycle Th17 for Inflammation (NEW!) Wnt Signaling Human miRNA Array (NEW!) Common Cytokines Notch Signaling Breast Cancer & Estrogen Receptor Chemokines TGFβ / BMP Signaling Tumor Metastasis NF-κB Signaling Pathway Endothelial Cell Biology Cancer PathwayFinder Th1-Th2-Th3 Osteogenesis Angiogenesis TNF Ligands Growth Factors Cancer Drug Resistance Toll-like Receptors ECM & Adhesion Signal Transduction Toxicology & Drug Metabolism Neuroscience Signal Transduction PathwayFinder Drug Metabolism Neuroscience Ion Channels NFkB Signaling Drug Phase I Enzymes Neurotransmitter Receptors Jak / Stat Signaling Drug Transporters Neurotrophins & Receptors DNA Damage Signaling Oxidative Stress Neurogenesis and Neural Stem Cell Insulin Signaling Stress & Toxicity MAP Kinase Signaling Other Diseases Custom PCR Arrays cAMP / Calcium Signaling Atherosclerosis 96-Well Custom Arrays p53 Signaling Diabetes 384-Well Custom Arrays Complete PCR Array List For Internal Use Only -7- Sample & Assay Technologies
- 8. Accessing PCR Array Content For Internal Use Only -8- Sample & Assay Technologies
- 9. PCR Array: Functional Gene Groupings For Internal Use Only -9- Sample & Assay Technologies
- 10. PCR Array Layout For Internal Use Only - 10 - Sample & Assay Technologies
- 11. miScript miRNA PCR Arrays Complete miRNA genome (miRNome) miScript miRNA Pathway Arrays Human miScript miRNA PCR Array Mouse miScript miRNA PCR Array Rat miScript miRNA PCR Array Dog miScript miRNA PCR Array Species Number of assays Human, Mouse, Rat: Brain Cancer Cancer Cell Differentiation & Development Immunopathology Inflammation miFinder Neurological Development & Disease Serum & Plasma Total Number of Plates (miRBase V16) 96 well format 384 well format Human 1068 12 3 Mouse 947 12 3 Rat 653 8 2 Dog 277 4 1 For Internal Use Only - 11 - Sample & Assay Technologies
- 12. Custom PCR Arrays – VERY POPULAR! Analyze the Genes Most Important To 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 For Internal Use Only - 12 - Sample & Assay Technologies
- 13. PCR Array Species Expansion 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 For Internal Use Only - 13 - Sample & Assay Technologies
- 14. Wet-Bench Validation of ALL qPCR Assays • Sensitivity Constant C(t) value for same amount of template • Amplification Efficiency DART-PCR analysis • Specificity • Single, symmetric dissociation curves • Single band of appropriate size on agarose gel • Dynamic Range Linear over 8 logs of template • NTC No primer dimer formation For Internal Use Only - 14 - Sample & Assay Technologies
- 15. Exceptional Specificity • 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. CONCLUSION: Each well on PCR Arrays detects a single gene-specific product. For Internal Use Only - 15 - Sample & Assay Technologies
- 16. Excellent Reproducibility Reproducibility Among Different Users 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. For Internal Use Only - 16 - Sample & Assay Technologies
- 17. Application Example: miRNA Profiling For Internal Use Only - 17 - Sample & Assay Technologies
- 18. Application Example: miRNA Profiling For Internal Use Only - 18 - Sample & Assay Technologies
- 19. 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 Arrays http://www.sabiosciences.com/support_publication.php For Internal Use Only - 19 - Sample & Assay Technologies
- 20. 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 For Internal Use Only - 20 - Sample & Assay Technologies
- 21. Cignal Reporter Assays (45 Pathways) Pathway Amino Acid Deprivation Androgen Antioxidant Response ATF6 C/EBP cAMP/PKA Cell Cycle DNA Damage EGR1 ER Stress Estrogen GATA Glucocorticoid Heat Shock Response Heavy Metal Stress Hedgehog HNF4 Hypoxia Interferon Regulation Type I Interferon Interferon Gamma KLF4 Liver X For Internal Use Only Pathway MAPK/ERK MAPK/JNK MEF2 c-myc Nanog NFkB Notch Oct4 Pax6 PI3K/AKT PKC/Ca++ PPAR Progesterone Retinoic Acid Retinoid X Sox2 SP1 STAT3 TGF-beta VDRE Wnt Xenobiotic Transcription Factor ATF4/ATF3/ATF2 Androgen Receptor Nrf2 & Nrf1 ATF6 C/EBP CREB E2F/DP1 p53 EGR1 CBF/NF-Y/YY1 ER GATA GR HSF MTF1 Gli HNF4 HIF-1 IRF-1 STAT1/STAT2 STAT1/STAT1 KLF4 LXR - 21 - Transcription Factor Elk-1/SRF AP-1 MEF2 Myc/Max Nanog NFkB RBP-Jk Oct4 Pax6 FOXO NFAT PPAR PR RAR RXR Sox2 SP1 STAT3 SMAD2/3/4 Vitamin D Receptor TCF/LEF AhR Sample & Assay Technologies
- 22. Cignal Reporter Assays: Experimental Design For Internal Use Only - 22 - Sample & Assay Technologies
- 23. Cignal Finder Arrays For Internal Use Only - 23 - Sample & Assay Technologies
- 24. How Does a Cignal Finder Array Work? For Internal Use Only - 24 - Sample & Assay Technologies
- 25. Application Example: Signal Transduction J. Biol. Chem. 2006;281: 24085-9. For Internal Use Only - 25 - Sample & Assay Technologies
- 26. Application Example: Signal Transduction For Internal Use Only - 26 - Sample & Assay Technologies
- 27. Application Example: Signal Transduction For Internal Use Only - 27 - Sample & Assay Technologies
- 28. 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 For Internal Use Only - 28 - Sample & Assay Technologies
- 29. 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 Researchers How do I use the Cignal Reporters in cells that are difficult or impossible to transfect? For Internal Use Only - 29 - Sample & Assay Technologies
- 30. Cignal Pathway Reporter System Products Primary cells, stem cells, and difficult to transfect cell lines Easy to transfect cell lines Endpoint Assays Cignal Dual-Luciferase Reporter Assays Single Pathway Assays For Internal Use Only 10-Pathway Arrays Dynamic Live Cell Assays Dynamic Live Cell Assays Endpoint Assays Cignal GFP Reporter Assays Cignal Lenti Luciferase Reporters Single Pathway Assays Single Pathway Assays - 30 - 10-Pathway Arrays Cignal Lenti GFP Reporters Single Pathway Assays Sample & Assay Technologies
- 31. Cignal Lenti Reporter Assays (35 Pathways) Pathway Amino Acid Deprivation Androgen Antioxidant Response ATF6 C/EBP cAMP/PKA Cell Cycle DNA Damage EGR1 ER Stress Estrogen GATA Glucocorticoid Heat Shock Response Heavy Metal Stress Hedgehog HNF4 Hypoxia Interferon Regulation Type I Interferon Interferon Gamma KLF4 Liver X For Internal Use Only Pathway MAPK/ERK MAPK/JNK MEF2 c-myc Nanog NFkB Notch Oct4 Pax6 PI3K/AKT PKC/Ca++ PPAR Progesterone Retinoic Acid Retinoid X Sox2 SP1 STAT3 TGFβ VDRE Wnt Xenobiotic Transcription Factor ATF4/ATF3/ATF2 Androgen Receptor Nrf2 & Nrf1 ATF6 C/EBP CREB E2F/DP1 p53 EGR1 CBF/NF-Y/YY1 ER GATA GR HSF MTF1 Gli HNF4 HIF-1 IRF-1 STAT1/STAT2 STAT1/STAT1 KLF4 LXR - 31 - Transcription Factor Elk-1/SRF AP-1 MEF2 Myc/Max Nanog NFkB RBP-Jk Oct4 Pax6 FOXO NFAT PPAR PR RAR RXR Sox2 SP1 STAT3 SMAD2/3/4 Vitamin D Receptor TCF/LEF AhR Sample & Assay Technologies
- 32. 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 For Internal Use Only - 32 - Sample & Assay Technologies
- 33. Lenti Reporters: Challenging Cell Types PASMC (Normal Human Pulmonary Artery Smooth Muscle cells) RAW 264.7 (Mouse leukaemic monocyte macrophage cell line) NFkB Reporter NFAT Reporter PMA + Ionomycin Vehicle Vehicle C6 (Rat glioma cells) CV 1 (Normal African green monkey kidney fibroblast) RBP-Jk Reporter SRE Reporter Vehicle Ad-Neg For Internal Use Only LPS PMA + FBS Ad-NICD - 33 - Sample & Assay Technologies
- 34. NFkB Pathway Sensor Cell Line Lenti Reporters Enable Rapid Pathway Sensor Cell Line Generation For Internal Use Only - 34 - Sample & Assay Technologies
- 35. Summary: Cignal Lenti Reporters . Breadth of Pathways and Arrays • 35 Pathway-Focused Reporters • 2 application-focused Cignal Finder Lenti 10-Pathway Arrays . Performance Exceptional sensitivity, specificity, signal-to-noise ratio, and reproducibility, using either luciferase or GFP reporter 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 For Internal Use Only - 35 - Sample & Assay Technologies
- 36. Take Home Message Tell a better Biological Story from your experiments • Publish faster and in higher impact journals • Work SMARTER, NOT Harder • Improve Success Rates of Your Grant Applications • Expedite Drug Pipeline For Internal Use Only - 36 - Sample & Assay Technologies
- 37. Webinar QUESTIONS? Louis Lichten 661.702.3133 Louis.Lichten@qiagen.com For Internal Use Only - 37 - Sample & Assay Technologies