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Cignal reporter assays webinar 2013

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  • 1. Cignal™ Reporter Assays Functional Analysis of Genes Biologics and Small Molecule Compounds Krishnan Allampallam, Ph.D.,MBA PCR Array & Pathway Signaling The Cignal Reporter Assays are intended for molecular biology applications. This product is not intended for the diagnosis, prevention or treatment of a disease. For Internal Use Only -1- Sample & Assay Technologies
  • 2. Topics to be covered ■ Introduction ■ What are Cignal Reporter Assays ■ How Cignal Reporter Assays work ■ How you can use Cignal Reporter Assays in your research For Internal Use Only -2- Sample & Assay Technologies
  • 3. Research challenge Post-Genomics Moving from a Signaling Flowchart Perspective to a Signaling Network Perspective. For Internal Use Only -3- Sample & Assay Technologies
  • 4. Research challenge Constructing Signaling Networks For Internal Use Only -4- Sample & Assay Technologies
  • 5. Research challenge Constructing signaling networks For Internal Use Only -5- Sample & Assay Technologies
  • 6. Cell-based assays Research Challenges ■ Intra-experiment variability Well-to-well, plate-to-plate reproducibility ■ Inter-experiment variability “status” of the cells who in the lab is conducting the experiments. ■ Assay performance Sensitivity, specificity, signal to noise ratio, and simplicity For Internal Use Only -6- Sample & Assay Technologies
  • 7. Cignal™ Reporter Assays Flexible, pre-optimized solution for cell based assays ■ Functionally validated dual-reporter formulation (Luciferase) Minimizes experimental variability, increasing biological relevance ■ Ready-to-use reporters and controls, in transfection ready format Enables simple, rapid analysis of the regulation of 45 signal transduction pathways ■ Genetically engineered transcriptional regulatory elements (TREs) & Destabilized , codon –optimized firefly luciferase Increases the signal to noise ratio, maximizes assay sensitivity and specificity Available in both Luciferase and GFP formats For Internal Use Only -7- Sample & Assay Technologies
  • 8. Topics to be covered ■ Introduction ■ What are Cignal Reporter Assays Challenges associated Solution: Cignal reporter Assay ■ How Cignal Reporter Assays work Complete Product Portfolio Dual-Luciferase Assays High performace Luciferase Pre-optimized TREs ■ How you can use Cignal Reporter Assays in your research For Internal Use Only -8- Sample & Assay Technologies
  • 9. Cignal™ Reporter AssaysComplete Solution Cignal Reporter Assays: Flexible solution: Multiple Reporter Formats Pathway-targeted Transcriptional Regulatory Elements (TRE), which establish the pathway specificity of each reporter TATA box Reporter Construct GFP/firefly luciferase Tandem repeats of TRE For Internal Use Only -9- Reporter Protein is expressed when TF binds to TRE Sample & Assay Technologies
  • 10. Cignal™ Reporter Assays Flexible solution: Multiple Reporter Formats Pathway-targeted Transcriptional Regulatory Elements (TRE), which establish the pathway specificity of each reporter TATA box Reporter Construct GFP/firefly luciferase Tandem repeats of TRE EGFP TF FL Upstream Signaling Events For Internal Use Only - 10 - Sample & Assay Technologies
  • 11. Cignal™ Reporter Assays Multiple Reporter Formats – Complete dual luciferase system Pathway-targeted Transcriptional Regulatory Elements (TRE), which establish the pathway specificity of each reporter TATA box Reporter Construct firefly luciferase Tandem repeats of TRE TATA box Negative Control Construct firefly luciferase firefly luciferase Positive Control Construct CMV TATA box Internal Control Construct CMV TATA box For Internal Use Only - 11 - Renilla Luciferase Sample & Assay Technologies
  • 12. Cignal™ Reporter Assays Plasmid - 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 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 - 12 - Pathway MAPK/ERK MAPK/JNK MEF2 c-myc Nanog NFκB Notch Oct4 Pax6 PI3K/AKT PKC/Ca++ PPAR Progesterone Retinoic Acid Retinoid X Sox2 SP1 STAT3 TGFβ VDRE Wnt Xenobiotic Transcription Factor Elk-1/SRF AP-1 MEF2 Myc/Max Nanog NFκB 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
  • 13. Cignal™ Reporter Assays Product offering 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 10-Pathway Arrays Dynamic Live Cell Assays Cignal GFP Reporter Assays For Internal Use Only Cignal Lenti Luciferase Reporters Single Pathway Assays Multiple options to suit your research needs: Dynamic Live Cell Assays Endpoint Assays Single Pathway Assays 10-Pathway Arrays Cignal Lenti GFP Reporters Single Pathway Assays Plasmid and Lentiviral, Luciferase and GFP reporters, Single, 10-pathway, 45-pathway - 13 - Sample & Assay Technologies
  • 14. Cignal™ Reporter Assays Lentiviral 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 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 - 14 - Pathway MAPK/ERK MAPK/JNK MEF2 c-myc Nanog NFκB Notch Oct4 Pax6 PI3K/AKT PKC/Ca++ PPAR Progesterone Retinoic Acid Retinoid X Sox2 SP1 STAT3 TGFβ VDRE Wnt Xenobiotic Transcription Factor Elk-1/SRF AP-1 MEF2 Myc/Max Nanog NFκB 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
  • 15. Using Cignal™ Reporter Assays What you need • Cignal Reporter Assay • Reliable transfection reagent and protocol (Attractene, HiPerFect) • Test Biological •shRNA (SureSilencing shRNA) • expression vectors (QIAgenes) • protein/peptide • small molecule • Detection Instrument and Luciferase Substrates For Internal Use Only - 15 - Sample & Assay Technologies
  • 16. Cignal™ Reporter Assays Key advantages – Assay design- Dual Luciferase Pathway-targeted transcriptional regulatory elements (45) Reporter Construct TRE TATA box Firefly luciferase Normalization Construct CMV TATA box Renilla luciferase Constitutive transcriptional regulatory element (1) The Transcriptional Regulatory Elements (TREs) establishes the pathway specificity of each reporter! For Internal Use Only - 16 - Sample & Assay Technologies
  • 17. Cignal™ Reporter Assays Why Dual Luciferase Sources of Variability in Cell-based Assays: • Number of cells seeded per well • Transfection efficiencies within and across plates • Multichannel pipettor inconsistencies • “Edge effects” influencing cell culture • Viability of cells following transfection/treatment • Lysis efficiencies within and across plates • Variability inherent to reporter assay Dual-Luciferase Assay design corrects for these unwanted sources of variability. • Changes due to the designed treatments of cells • RNA interference • Overexpression • Recombinant protein/peptide/growth factor • Small molecule For Internal Use Only - 17 - Sample & Assay Technologies
  • 18. Cignal™ Reporter Assays Dual Luciferase Assays - data Dual-Luciferase Assays: NFkB Reporter Assay Single-Luciferase Assays: NFkB Reporter Assay 30000000 2.5 CV=0.07 Luminescence (Relative Light Units) Relative Luciferase Units CV=0.70 25000000 20000000 15000000 CV=0.69 10000000 5000000 0 2 1.5 1 CV=0.09 0.5 0 NFkB Reporter + NFkB siRNA NFkB Reporter + Neg. control siRNA NFkB Reporter + NFkB siRNA NFkB Reporter + Neg. control siRNA Dual Luciferase Format Minimizes Variability For Internal Use Only - 18 - Sample & Assay Technologies
  • 19. Cignal™ Reporter Assays Advantage – Genetic engineered luciferase Mammalian codon-optimized AND Destabilized firefly luciferase Reporter Construct TRE TATA box Firefly luciferase Normalization Construct CMV TATA box Renilla luciferase For Internal Use Only - 19 - Sample & Assay Technologies
  • 20. Cignal™ Reporter Assays Advantage – Genetic engineered luciferase Modified Luciferase: NFkB Reporter Signal Following TNFα Induction α Modified Luciferase: Background NFkB Reporter Signal (no treatment post-transfection) 250 Relative Luciferase Units 0.35 Fold Induction 200 150 WHY? 100 50 0.3 0.25 0.2 0.15 0.1 0.05 0 0 Negative Control NFkB Reporter: NFkB Reporter: Stable Luc Destabilized Luc Negative Control NFkB Reporter: NFkB Reporter: Stable Luc Destabilized Luc High Signal to Noise Ratio Offers higher sensitivity For Internal Use Only - 20 - Sample & Assay Technologies
  • 21. Cignal™ Reporter Assays Advantage – Genetic engineered TRE, optimized for each pathway Pathway-targeted transcriptional regulatory elements (45) Reporter Construct TRE TATA box Firefly luciferase Normalization Construct CMV TATA box Renilla luciferase For Internal Use Only - 21 - Sample & Assay Technologies
  • 22. Cignal™ Reporter Assays Advantage – Genetic engineered TRE, optimized for each pathway Dual-Luciferase Assays Utilizing Cignal p53 TRE Dual-Luciferase Assays Utilizing Common p53 TRE 160 3 140 Relative Luciferase Units Relative Luciferase Units 3.5 2.5 2 2.5-Fold 1.5 1 0.5 0 120 100 80 60 125-Fold 40 20 0 p53 Reporter + DMSO p53 Reporter + 1µM Doxorubicin p53 Reporter + DMSO p53 Reporter + 1µM Doxorubicin TRE Maximizes Luciferase Signal For Internal Use Only - 22 - Sample & Assay Technologies
  • 23. Cignal™ Reporter Assays Experimental Design For Internal Use Only - 23 - Sample & Assay Technologies
  • 24. Cignal™ Reporter Assays Complete GFP system: to study interactions in individual cells Pathway-targeted Transcriptional Regulatory Elements (TRE), which establish the pathway specificity of each reporter TATA box GFP TATA box Reporter Construct GFP TATA box GFP Tandem repeats of TRE Negative Control Construct Positive Control Construct For Internal Use Only CMV - 24 - Sample & Assay Technologies
  • 25. Cignal™ Reporter Assays DNA-Vector GFP – 16 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 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 - 25 - Pathway MAPK/ERK MAPK/JNK MEF2 c-myc Nanog NFκB Notch Oct4 Pax6 PI3K/AKT PKC/Ca++ PPAR Progesterone Retinoic Acid Retinoid X Sox2 SP1 STAT3 TGFβ VDRE Wnt Xenobiotic Transcription Factor Elk-1/SRF AP-1 MEF2 Myc/Max Nanog NFκB 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
  • 26. Cignal™ Reporter Assays Lenti- GFP Reporter – 6 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 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 - 26 - Pathway MAPK/ERK MAPK/JNK MEF2 c-myc Nanog NFκB Notch Oct4 Pax6 PI3K/AKT PKC/Ca++ PPAR Progesterone Retinoic Acid Retinoid X Sox2 SP1 STAT3 TGFβ VDRE Wnt Xenobiotic Transcription Factor Elk-1/SRF AP-1 MEF2 Myc/Max Nanog NFκB 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
  • 27. Cignal™ Reporter Assays GFP system application: Small molecule studies Fluorometry Fluorescent Microscopy Treatment of Cignal SRE (GFP) transfectants with 10% serum and 10 ng/ml PMA activates the MAPK/ERK signaling pathway. For Internal Use Only - 27 - Sample & Assay Technologies
  • 28. Topics to be covered ■ Introduction to SABiosciences ■ What are Cignal Reporter Assays Challenges associated Solution: Cignal reporter Assay ■ How Cignal Reporter Assays work Complete Product Portfolio Dual-Luciferase Assays High performace Luciferase Pre-optimized TREs ■ How you can use Cignal Reporter Assays in your research For Internal Use Only - 28 - Sample & Assay Technologies
  • 29. Cignal™ Reporter Assays Application in multiple research areas Functional Genomics What’s the phenotype of my gene? Does my gene of interest regulate signaling pathway “X”? Functional Proteomics What’s the phenotype of my protein/peptide? Does my protein, peptide, or growth factor of interest regulate signaling pathway “X”? Drug Screening What is the mechanism of action of my small molecule drug candidates? Do my drug candidates of interest regulate signaling pathway “X”? For Internal Use Only - 29 - Sample & Assay Technologies
  • 30. Cignal™ Reporter Assays Application – RNA Interference For Internal Use Only - 30 - Sample & Assay Technologies
  • 31. Cignal™ Reporter Assays Application in multiple research areas Functional Genomics What’s the phenotype of my gene? Does my gene of interest regulate signaling pathway “X”? Functional Proteomics What’s the phenotype of my protein/peptide? Does my protein, peptide, or growth factor of interest regulate signaling pathway “X”? Drug Screening What is the mechanism of action of my small molecule drug candidates? Do my drug candidates of interest regulate signaling pathway “X”? For Internal Use Only - 31 - Sample & Assay Technologies
  • 32. Cignal™ Reporter Assays Application – Functional Proteomics For Internal Use Only - 32 - Sample & Assay Technologies
  • 33. Cignal™ Reporter Assays Application in multiple research areas Functional Genomics What’s the phenotype of my gene? Does my gene of interest regulate signaling pathway “X”? Functional Proteomics What’s the phenotype of my protein/peptide? Does my protein, peptide, or growth factor of interest regulate signaling pathway “X”? Drug Screening What is the mechanism of action of my small molecule drug candidates? Do my drug candidates of interest regulate signaling pathway “X”? For Internal Use Only - 33 - Sample & Assay Technologies
  • 34. Cignal™ Reporter Assays Application – Small Molecule Studies For Internal Use Only - 34 - Sample & Assay Technologies
  • 35. Cignal reporter assays Research challenge Application – Study pathway interaction Pathway Interaction Study Situation I have a biological molecule that knocks down p53 expression Question 1. What is impact of p53 gene expression knock down on the p53 signaling pathway? 2. What is the impact of p53 knock down on other cancerrelevant signaling pathways? For Internal Use Only - 35 - Sample & Assay Technologies
  • 36. Research challenge Pathway interactions Wnt Signaling NFkB Signaling p53 Signaling ? ? E2F Signaling ? ? Hypoxia Signaling MAPK/JNK Signaling ? ? ? TGFβ Signaling MAPK/ERK Signaling ? ? Myc Signaling For Internal Use Only Notch Signaling - 36 - Sample & Assay Technologies
  • 37. Cignal™ Reporter Assays Application – study pathway interaction Approach: Carry out a p53 RNA interference experiment with multiple cancer-relevant Cignal Reporter Assays For Internal Use Only - 37 - Sample & Assay Technologies
  • 38. Cignal Finder arrays Cancer array design Pathway Cell Cycle DNA Damage Hypoxia MAPK/ERK MAPK/JNK c-Myc NFkB Notch TGFβ Wnt For Internal Use Only Transcription Factor E2F/DP1 p53 HIF Elk-1/SRF AP1 Myc/Max NFkB RBP-Jk SMAD2/SMAD3/SMAD4 TCF/LEF - 38 - Sample & Assay Technologies
  • 39. Cignal Finder array How does it work? For Internal Use Only - 39 - Sample & Assay Technologies
  • 40. Cignal Finder cancer array p53 RNAi study design 1. p53 Cignal Reporter 2. RBP-Jk Cignal Reporter 3. TCF/LEF Cignal Reporter 4. E2F Cignal Reporter 5. AP1 Cignal Reporter 6. SRE Cignal Reporter 7. SMAD Cignal Reporter 8. NFkB Cignal Reporter 9. Myc Cignal Reporter 1 2 4 3 5 6 7 8 9 10 11 12 p53 siRNA Neg. control siRNA 10. HIF Cignal Reporter 11. Cignal Negative Control 12. Cignal Positive Control For Internal Use Only - 40 - Sample & Assay Technologies
  • 41. Cignal Finder cancer array p53 RNAi study design - results Impact of p53 siRNA Treatment For Internal Use Only - 41 - Sample & Assay Technologies
  • 42. Cignal Finder cancer array p53 RNAi study design – next steps p53 Signaling Hypoxia Signaling MAPK/ERK Signaling Notch Signaling For Internal Use Only - 42 - Sample & Assay Technologies
  • 43. Cignal™ Reporter Assays System Summary ■ Breadth of Pathways and Research Application ■ 45 Pathway-Focused Reporter Assays ■ 6 Application-Specific Cignal Finder 10-Pathway Arrays ■ 1 45-Pathway Reporter Array ■ Performance ■ Exceptional Sensitivity, Specificity, Signal-to-Noise Ratio, and Reproducibility ■ Convenience ■ Ready-to-use, validated, preformatted transient assays ■ Dual Modalities ■ Dual-luciferase quantitative endpoint assays ■ GFP dynamic live cell assays with single cell resolution For Internal Use Only - 43 - Sample & Assay Technologies
  • 44. New research tool for 2013 http://www.sabiosciences.com/sciglobe.php Sample & Assay Technologies
  • 45. Cignal™ Reporter Assays For rapidly analyzing pathway signaling activity Questions? Contact Technical Support 9 AM – 6 PM Eastern M – F Telephone: 888-503-3187 Email: support@SABiosciences.com Thank you! Krishnan Allampallam QIAwebinars@qiagen.com For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at www.qiagen.com or can be requested from QIAGEN Technical Services or your local distributor For Internal Use Only - 45 - Sample & Assay Technologies