Cell-based Reporter Assays: Measure 45 Signaling Pathway Activity in Any Cell Type

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qiagen
PowerPoint Presentation Contact: Anisha Kharkia; anisha.kharkia@qiagen.com Cignal Plasmid and Lenti Reporter Kits Sample to Insight 1 Cignal Plasmid and Lenti Reporter Kits Functional analysis of genes, biologics and small molecule compounds  The Cignal Reporter Assays are intended for molecular biology applications. These products are not intended for the diagnosis, prevention or treatment of a disease. Sample to Insight How you can use reporter assays in your research 3 Taking on challenges with reporter and lenti assays 1 How Cignal Reporter and Lenti Assays work 2 Agenda 3 Summary 4 Image reference: Apoptosis through death receptors. Pathway central, GeneGlobe. Sample to Insight 3 How you can use Cignal Reporter Assays in your research 3 Taking on challenges with reporter and lenti assays 1 How Cignal Reporter and Lenti Assays work 2 Agenda 4 Summary 4 Challenges associated a Solution: Cignal Reporter and Lenti Assay b Image reference: Apoptosis through death receptors. Pathway central, GeneGlobe. Sample to Insight 4 Research challenge: post-genomics Moving from a signaling flowchart perspective to a signaling network perspective DNA damage ATM/ATR p53 Cell cycle arrest/ apoptosis Basic signal transduction flowchart Sample to Insight 5 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 Sample to Insight Cell-based signaling studies: the challenges Frustration of analyzing one pathway at a time Time consuming Misleading Studies limited to cells that are easily transfected What about primary cells? What about stem cells? What about cell lines that are difficult to transfect? Sample to Insight 7 Solutions provided by Cignal Lenti Reporter Assays Engineered and formatted for superior performance Custom engineered transcriptional response elements (TRE) Use reporter genes with outstanding sensitivity and specificity Internal, positive and negative controls for reproducible results Luciferase and GFP formats are available Lentiviral particles are ready for transduction right out of the box Cignal Finder 10-Pathway Arrays Enable the rapid and reliable study of multiple signaling pathways Sample to Insight 8 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 to Insight Research challenge: constructing signaling networks Image reference: TNF pathway and interaction network. GeneGlobe Sample to Insight Research challenge: constructing signaling networks A B C D Signal transduction cascade (~40 proteins) Image reference: SMAD signaling network Sample to Insight 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) and destabilized, codon-optimized firefly luciferase Increases the signal to noise ratio, maximizes assay sensitivity and specificity Available in both luciferase and GFP formats Sample to Insight Agenda 13 How you can use Cignal Reporter Assays in your research 3 Taking on challenges with Cignal Reporter Assays 1 How Cignal Reporter and Lenti Assays work 2 Summary 4 Image reference: Apoptosis through death receptors. Pathway central, GeneGlobe. Sample to Insight 13 How you can use Cignal Reporter Assays in your research 3 Taking on challenges with Cignal Reporter Assays 1 How Cignal Reporter and Lenti Assays work 2 Agenda 14 Complete product portfolio a Dual-luciferase assays b Summary 4 High-performance luciferase a Pre-optimized TREs b Image reference: Apoptosis through death receptors. Pathway central, GeneGlobe. Sample to Insight 14 Two reporter modalities Dual-luciferase format Quantitative end-point luminescence assay Exceptional reproducibility, sensitivity and signal-to-noise ratio Average expression from a population of cells GFP format Dynamic live cell assay Single cell resolution Readout flexibility (flow cytometry, fluorescent microscopy, fluorometry) Sample to Insight 15 Flexible solution: multiple reporter formats Reporter protein is expressed when TF binds to TRE Reporter construct Pathway-targeted Transcriptional Regulatory Elements (TRE), which establish the pathway specificity of each reporter Sample to Insight 16 Reporter construct Tandem Repeats of TRE Pathway-targeted Transcriptional Regulatory Elements (TRE), which establish the pathway specificity of each reporter Upstream signaling events TF EGFP FL Flexible solution: multiple reporter formats Sample to Insight 17 Cignal Reporter and Lenti Reporter Assays: complete solution Rigorously optimized, functionally validated and ready-to-use reporters Positive control construct Negative control construct Internal control construct Reporter construct Pathway-targeted Transcriptional Regulatory Elements (TRE), which establish the pathway specificity of each reporter Sample to Insight 18 Positive control construct Negative control construct Internal control construct Multiple reporter formats: complete dual Luciferase system Reporter construct Pathway-targeted Transcriptional Regulatory Elements (TRE), which establish the pathway specificity of each reporter Sample to Insight 19 Easy-to- transfect cell lines Primary cells, stem cells and difficult-to-transfect cell lines End-point Assays Dynamic Live Cell Assays Dynamic Live Cell Assays Cignal Dual- Luciferace Reporter Assays Cignal GFP Reporter Assays Cignal Luc/ GFP Reporter Assays Cignal Lenti GFP / Luc Reporter Assays Single Pathway Assays 10-Pathway Arrays 45-Pathway Arrays Single Pathway Assays Single Pathway Assays Single Pathway Assays End-point Assays Cignal Lenti Luciferace Reporter Assays Cignal GFP Luciferase Reporter Assays 45-Pathway Arrays 10-Pathway Arrays Single Pathway Assays Single Pathway Assays Multiple options to suit your research needs: plasmid and lentiviral, luciferase and GFP reporters, single, 10-pathway, 45-pathway Cignal Reporter and Lenti Reporter Assays: product offering Sample to Insight Cignal Reporter Assays: Plasmid – 45 Pathways Pathway Transcription Factor Amino Acid Deprivation  ATF4/ATF3/ATF2  Androgen  Androgen Receptor  Antioxidant Response  Nrf2 & Nrf1  ATF6  ATF6  C/EBP C/EBP cAMP/PKA CREB Cell Cycle E2F/DP1 DNA Damage p53 EGR1  EGR1  ER Stress  CBF/NF-Y/YY1  Estrogen ER GATA GATA Glucocorticoid GR Heat Shock Response  HSF  Heavy Metal Stress  MTF1  Hedgehog Gli HNF4 HNF4 Hypoxia HIF-1 Interferon Regulation IRF-1 Type I Interferon STAT1/STAT2 Interferon Gamma STAT1/STAT1 KLF4 KLF4 Liver X LXR Pathway Transcription Factor MAPK/ERK Elk-1/SRF MAPK/JNK AP-1 MEF2  MEF2  c-myc Myc/Max Nanog Nanog NFκB NFκB  Notch RBP-Jk Oct4  Oct4  Pax6 Pax6 PI3K/AKT  FOXO  PKC/Ca++ NFAT PPAR  PPAR  Progesterone PR Retinoic Acid RAR Retinoid X RXR Sox2 Sox2 SP1  SP1  STAT3  STAT3  TGFβ SMAD2/3/4 VDRE  Vitamin D Receptor  Wnt TCF/LEF Xenobiotic AhR Sample to Insight Cignal Reporter Assays: Lentiviral Assays (35 Pathways) Pathway Transcription Factor Amino Acid Deprivation  ATF4/ATF3/ATF2  Androgen  Androgen Receptor  Antioxidant Response  Nrf2 & Nrf1  ATF6  ATF6  C/EBP C/EBP cAMP/PKA CREB Cell Cycle E2F/DP1 DNA Damage p53 EGR1  EGR1  ER Stress  CBF/NF-Y/YY1  Estrogen ER GATA GATA Glucocorticoid GR Heat Shock Response  HSF  Heavy Metal Stress  MTF1  Hedgehog Gli HNF4 HNF4 Hypoxia HIF-1 Interferon Regulation IRF-1 Type I Interferon STAT1/STAT2 Interferon Gamma STAT1/STAT1 KLF4 KLF4 Liver X LXR Pathway Transcription Factor MAPK/ERK Elk-1/SRF MAPK/JNK AP-1 MEF2  MEF2  c-myc Myc/Max Nanog Nanog NFκB NFκB  Notch RBP-Jk Oct4  Oct4  Pax6 Pax6 PI3K/AKT  FOXO  PKC/Ca++ NFAT PPAR  PPAR  Progesterone PR Retinoic Acid RAR Retinoid X RXR Sox2 Sox2 SP1  SP1  STAT3  STAT3  TGFβ SMAD2/3/4 VDRE  Vitamin D Receptor  Wnt TCF/LEF Xenobiotic AhR Sample to Insight 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 Sample to Insight Using Cignal Lenti Reporter Assay What you need: Cignal Lenti Reporter or 10-Pathway Array SureENTRY™ can enhance transduction efficiency in most cell types Test biological siRNA (Flexitube siRNA) shRNA (SureSilencing shRNA) expression vectors (Qiagenes) protein/peptide small molecule Luciferase Assay Kit (Promega) and luminometer or GFP detection system Sample to Insight 24 Cignal Reporter & Lenti Reporter Assays Reporter construct Normalization construct Pathway-targeted transcriptional regulatory elements (45) Constitutive transcriptional regulatory element (1) The Transcriptional Regulatory Elements (TREs) establishe the pathway specificity of each reporter! Key advantages, assay design, dual-luciferase Sample to Insight Cignal Reporter and Lenti 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 Changes due to the designed treatments of cells RNA interference Overexpression Recombinant protein/peptide/growth factor Small molecule Dual-luciferase assay design corrects for these unwanted sources of variability. Sample to Insight Dual-luciferase assays: NFkB Reporter Assay CV=0.09 CV=0.07 Single-luciferase assays: NFkB Reporter Assay CV=0.69 CV=0.70 NFkB Reporter + NFkB siRNA NFkB Reporter + Neg. control siRNA NFkB Reporter + NFkB siRNA NFkB Reporter + Neg. control siRNA Dual-luciferase format minimizes variability Relative Luciferase Units Luminescence (Relative Light Units) Sample to Insight Modified luciferase: background NFkB Reporter signal (no treatment post-transfection) Modified luciferase: NFkB Reporter signal following TNF induction WHY? Negative control NFkB Reporter: Stable Luc NFkB Reporter: Destabilized Luc Negative control NFkB Reporter: Stable Luc NFkB Reporter: Destabilized Luc Advantage: genetic engineered luciferase High signal-to-noise ratio offers higher sensitivity Relative Luciferase Units Fold Induction Sample to Insight Dual-luciferase assays utilizing Cignal p53 TRE p53 Reporter + DMSO p53 Reporter + 1µM Doxorubicin Dual-luciferase assays utilizing common p53 TRE p53 Reporter + DMSO p53 Reporter + 1µM Doxorubicin 125-Fold 2.5-Fold TRE maximizes luciferase signal Relative Luciferase Units Relative Luciferase Units Sample to Insight Cignal Reporter and Lenti Reporter optimization process Improved sensitivity and biological relevance: Experimentally optimized response elements : Sequence of transcription response element (TRE) Number of TREs Intervening sequence between TRE Engineered reporter genes: Destabilized firefly luciferase (Maximize signal-to-noise ratio) Codon optimization for mammalian expression (Maximize expression) Removal of hidden transcription factor binding sites (Reduce background) Maximize specificity and sensitivity Save time, money and labor: use Cignal Reporters and Lenti Reporters Sample to Insight 30 Cignal Reporter Assays Pathway Transcription Factor Amino Acid Deprivation  ATF4/ATF3/ATF2  Androgen  Androgen Receptor  Antioxidant Response  Nrf2 & Nrf1  ATF6  ATF6  C/EBP C/EBP cAMP/PKA CREB Cell Cycle E2F/DP1 DNA Damage p53 EGR1  EGR1  ER Stress  CBF/NF-Y/YY1  Estrogen ER GATA GATA Glucocorticoid GR Heat Shock Response  HSF  Heavy Metal Stress  MTF1  Hedgehog Gli HNF4 HNF4 Hypoxia HIF-1 Interferon Regulation IRF-1 Type I Interferon STAT1/STAT2 Interferon Gamma STAT1/STAT1 KLF4 KLF4 Liver X LXR Pathway Transcription Factor MAPK/ERK Elk-1/SRF MAPK/JNK AP-1 MEF2  MEF2  c-myc Myc/Max Nanog Nanog NFκB NFκB  Notch RBP-Jk Oct4  Oct4  Pax6 Pax6 PI3K/AKT  FOXO  PKC/Ca++ NFAT PPAR  PPAR  Progesterone PR Retinoic Acid RAR Retinoid X RXR Sox2 Sox2 SP1  SP1  STAT3  STAT3  TGFβ SMAD2/3/4 VDRE  Vitamin D Receptor  Wnt TCF/LEF Xenobiotic AhR DNA-vector GFP – 16 pathways Sample to Insight Cignal Lenti Reporter Assays Pathway Transcription Factor Amino Acid Deprivation  ATF4/ATF3/ATF2  Androgen  Androgen Receptor  Antioxidant Response  Nrf2 & Nrf1  ATF6  ATF6  C/EBP C/EBP cAMP/PKA CREB Cell Cycle E2F/DP1 DNA Damage p53 EGR1  EGR1  ER Stress  CBF/NF-Y/YY1  Estrogen ER GATA GATA Glucocorticoid GR Heat Shock Response  HSF  Heavy Metal Stress  MTF1  Hedgehog Gli HNF4 HNF4 Hypoxia HIF-1 Interferon Regulation IRF-1 Type I Interferon STAT1/STAT2 Interferon Gamma STAT1/STAT1 KLF4 KLF4 Liver X LXR Pathway Transcription Factor MAPK/ERK Elk-1/SRF MAPK/JNK AP-1 MEF2  MEF2  c-myc Myc/Max Nanog Nanog NFκB NFκB  Notch RBP-Jk Oct4  Oct4  Pax6 Pax6 PI3K/AKT  FOXO  PKC/Ca++ NFAT PPAR  PPAR  Progesterone PR Retinoic Acid RAR Retinoid X RXR Sox2 Sox2 SP1  SP1  STAT3  STAT3  TGFβ SMAD2/3/4 VDRE  Vitamin D Receptor  Wnt TCF/LEF Xenobiotic AhR Lenti-GFP Reporter – 6 pathways Sample to Insight Treatment of Cignal SRE (GFP) transfectants with 10% serum and 10 ng/ml PMA activates the MAPK/ERK signaling pathway. Fluorometry Fluorescence microscopy Cignal Reporter Assays GFP system application: small molecule studies Sample to Insight Agenda 34 How you can use Cignal Reporter Assays in your research 3 Taking on challenges with Cignal Reporter Assays 1 How Cignal Reporter and Lenti Assays work 2 Summary 4 Image reference: Apoptosis through death receptors. Pathway central, GeneGlobe. Sample to Insight 34 Cignal Lenti Reporters: primary cells PKC/Ca++ signaling pathway studies in primary arteriole smooth muscle cells Cignal Lenti Reporter system makes this study possible Relative Luciferase Units Sample to Insight Cignal Lenti Reporters: challenging cell lines NFkB signaling pathway studies in D1 Murine T-cell Leukemia cell line Cignal Lenti Reporter system makes this study possible Sample to Insight Cignal Lenti Reporters: challenging cell lines Cignal Lenti SRE Reporter (GFP) measures MAPK/ERK pathway signaling activity in African green monkey CV1 fibroblasts Cignal Lenti Reporter system makes this study possible Sample to Insight Cignal Lenti Reporters: sensor cell lines Generation of an NFkB signaling pathway stable sensor cell line Cignal Lenti Reporters enable rapid pathway sensor cell line generation P1 P5 P10 P15 Relative Luciferase Units Sample to Insight 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”? Cignal Reporter Assays: application in multiple research areas Sample to Insight Cignal Reporter Assays: application – RNA interference Relative Luciferase Units Functional genomics What’s the phenotype of my gene? Does my gene of interest regulate signaling pathway “X”? Sample to Insight Cignal Reporter Assays: application Functional proteomics What’s the phenotype of my protein/peptide? Does my protein, peptide, or growth factor of interest regulate signaling pathway “X”? Sample to Insight Cignal Reporter Assays: application – small molecule studies Relative Luciferase Units Drug screening What is the mechanism of action of my small molecule drug candidates? Do my drug candidates of interest regulate signaling pathway “X”? Sample to Insight Situation: I have a biological molecule that knocks down p53 expression Questions: What is impact of p53 gene expression knock down on the p53 signaling pathway? What is the impact of p53 knock down on other cancer-relevant signaling pathways? Research challenge: pathway interaction study Sample to Insight Wnt signaling NFkB signaling E2F signaling MAPK/ERK signaling Myc signaling MAPK/JNK signaling TGFβ signaling Notch signaling ? ? ? ? ? ? ? ? ? Hypoxia signaling p53 signaling Research challenge: pathway interactions Image reference: p53signaling. Pathway central, GeneGlobe. Sample to Insight Approach: Carry out a p53 RNA interference experiment with multiple cancer-relevant Cignal Reporter Assays Cignal Reporter Assays: application – study pathway interaction Sample to Insight Cignal Finder Arrays: cancer array design Pathway Cell Cycle DNA Damage Hypoxia MAPK/ERK MAPK/JNK c-Myc NFkB Notch TGFβ Wnt Transcription Factor E2F/DP1 p53 HIF Elk-1/SRF AP1 Myc/Max NFkB RBP-Jk SMAD2/SMAD3/SMAD4 TCF/LEF Sample to Insight 46 Cignal Finder Array: how does it work? Sample to Insight Cignal Finder Cancer Array: p53 RNAi study design p53 Cignal Reporter RBP-Jk Cignal Reporter TCF/LEF Cignal Reporter E2F Cignal Reporter AP1 Cignal Reporter SRE Cignal Reporter SMAD Cignal Reporter 8. NFkB Cignal Reporter 9. Myc Cignal Reporter 10. HIF Cignal Reporter 11. Cignal Negative control 12. Cignal Positive control p53 siRNA Neg. control siRNA Sample to Insight Impact of p53 siRNA treatment Cignal Finder Cancer Array: p53 RNAi study design – results Fold Change in Pathway Activation Sample to Insight MAPK/ERK signaling Notch signaling Hypoxia signaling p53 signaling Cignal Finder Cancer Array: p53 RNAi study design – next steps Image reference: p53signaling. Pathway central, GeneGlobe. Sample to Insight Cignal Lenti Reporters: biosafety VSV-G pseudotyped lentiviral particles Deletion in U3 portion of 3’LTR results in self-inactivation (SIN), following transduction of target cell No structural or replication genes are expressed in transduced cells, making Cignal lentiviral vectors replication-defective No virulence genes (vpr, vif, vpu, nef) are present in the Cignal Lenti Reporters Experiments should be performed under Biosafety Level 2 (BSL-2) conditions See our Cignal Lenti Reporter web pages for useful links Sample to Insight Tips for a successful transduction Avoid freeze thaws Result in activity loss MOI dilution series Recommended range 5–50 Transduction reagent Sure ENTRY usually in the range of 4–8 μg/ml MOIs >50 should be split Sequential 4-hour transductions Sample to Insight Tips for making stable cell lines Kill curve for Puromycin Don’t guess 500–10,000 ng/ml MOI for stables Perform a transient study to find appropriate MOI Freeze cells down Renewable reagent Sample to Insight Agenda 54 How you can use Cignal Reporter Assays in your research 3 Taking on challenges with Cignal Reporter Assays 1 How Cignal Reporter and Lenti Assays work 2 Summary 4 Image reference: Apoptosis through death receptors. Pathway central, GeneGlobe. Sample to Insight 54 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 Final benefit to researchers: Ready-to-use, validated reporters Don’t have to produce or amplify lentivirus in your laboratory Quick generation of reporter cell lines Sample to Insight 55 Summary: Cignal Reporter Assays system Breadth of pathways and research application 45 pathway-focused reporter assays 6 application-specific Cignal Finder 10-Pathway Arrays 1 Cignal 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 end-point assays GFP dynamic live cell assays with single cell resolution Sample to Insight Cignal Reporter and Lenti Reporter Assays 57 Questions? Contact technical support 9 AM – 6 PM EST Telephone: 800-362-7737 Or Contact Anisha Kharkia Global Product Manager Email: Anisha.Kharkia@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. Sample to Insight 57 Thank you Sample to Insight Chart1 5727326 10223380 2003220 15737362 23907364 3733934 Sheet1 Luciferase Renilla ratio Average SD CV NFkB reporter + NFkB1 siRNA 5727326 6051066 0.946 0.8753333333 0.0783985544 0.09 10223380 11495530 0.889 2003220 2531686 0.791 NFkB reporter + negative control siRNA 15737362 6968666 2.258 2.1156666667 0.1477712196 0.07 23907364 12176456 1.963 3733934 1756248 2.126 NFkB reporter + NFkBIA shRNA 6573910 3317632 1.982 2.3169052671 0.2982141376 0.13 86515384 35828692 2.415 36551702 14313144 2.5537158014 NFkB reporter + Rel A shRNA 2223454 4943094 0.45 0.5517868727 0.0947267092 0.17 6903218 12143928 0.568 3329888 5224496 0.6373606181 NFkB reporter + negative control shRNA 47583702 47919378 0.993 1.1472143585 0.1922742808 0.17 31502328 29003080 1.086 20282686 14884812 1.3626430754 SD CV Luciferase Luciferase Luciferase Avg. Triplicate 4116117 0.69 NFkB reporter + NFkB1 siRNA 5727326 10223380 2003220 5984642 10147237 0.7 NFkB reporter + negative control siRNA 15737362 23907364 3733934 14459553 40384974 0.93 NFkB reporter + NFkBIA shRNA 6573910 86515384 36551702 43213665 2445849 0.59 NFkB reporter + Rel A shRNA 2223454 6903218 3329888 4152187 13722466 0.41 NFkB reporter + negative control shRNA 47583702 31502328 20282686 33122905 Luciferase/Renilla ratio NFkB reporter + NFkB1 siRNA 0.946 0.889 0.791 NFkB reporter + negative control siRNA 2.258 1.963 2.126 NFkB reporter + NFkBIA shRNA 1.982 2.415 2.5537158014 NFkB reporter + Rel A shRNA 0.45 0.568 0.6373606181 NFkB reporter + negative control shRNA 0.993 1.086 1.3626430754 Use of dual luciferase, which is inbuild in cignal reporter assay, reduce experimental variability and provide more realiable results Sheet1 Luminescence (Relative Light Units) Sheet2 Relative Luciferase Units Sheet3 Chart2 0.946 0.889 0.791 2.258 1.963 2.126 Sheet1 Luciferase Renilla ratio Average SD CV NFkB reporter + NFkB1 siRNA 5727326 6051066 0.946 0.8753333333 0.0783985544 0.09 10223380 11495530 0.889 2003220 2531686 0.791 NFkB reporter + negative control siRNA 15737362 6968666 2.258 2.1156666667 0.1477712196 0.07 23907364 12176456 1.963 3733934 1756248 2.126 NFkB reporter + NFkBIA shRNA 6573910 3317632 1.982 2.3169052671 0.2982141376 0.13 86515384 35828692 2.415 36551702 14313144 2.5537158014 NFkB reporter + Rel A shRNA 2223454 4943094 0.45 0.5517868727 0.0947267092 0.17 6903218 12143928 0.568 3329888 5224496 0.6373606181 NFkB reporter + negative control shRNA 47583702 47919378 0.993 1.1472143585 0.1922742808 0.17 31502328 29003080 1.086 20282686 14884812 1.3626430754 SD CV Luciferase Luciferase Luciferase Avg. Triplicate 4116117 0.69 NFkB reporter + NFkB1 siRNA 5727326 10223380 2003220 5984642 10147237 0.7 NFkB reporter + negative control siRNA 15737362 23907364 3733934 14459553 40384974 0.93 NFkB reporter + NFkBIA shRNA 6573910 86515384 36551702 43213665 2445849 0.59 NFkB reporter + Rel A shRNA 2223454 6903218 3329888 4152187 13722466 0.41 NFkB reporter + negative control shRNA 47583702 31502328 20282686 33122905 Luciferase/Renilla ratio NFkB reporter + NFkB1 siRNA 0.946 0.889 0.791 NFkB reporter + negative control siRNA 2.258 1.963 2.126 NFkB reporter + NFkBIA shRNA 1.982 2.415 2.5537158014 NFkB reporter + Rel A shRNA 0.45 0.568 0.6373606181 NFkB reporter + negative control shRNA 0.993 1.086 1.3626430754 Use of dual luciferase, which is inbuild in cignal reporter assay, reduce experimental variability and provide more realiable results Sheet1 Luminescence (Relative Light Units) Sheet2 Relative Luciferase Units Sheet3 Chart1 0.0015676137 0.0001536241 0.2884662746 0.0409162997 0.0726666667 0.0005773503 Sheet1 Luc/renilla Ratio average SD fold change NFkB reporter with stable luciferase gene (TNF 50ng/ml) 8.2896305439 8.2857009402 0.0055572989 28.723291658 0.0192649866 8.2817713365 NFkB reporter with stable luciferase gene (Without TNF) 0.3173984677 0.2884662746 0.0409162997 0.2595340816 Negative control (TNF 50ng/ml) 0.0018535114 0.0017061745 0.0001524078 1.0883896197 0.0972227921 0.0015491573 0.0017158547 Negative control 0.0016857211 0.0015676137 0.0001536241 0.0013939373 0.0016231827 NFkB reporter with desrabilized luciferase gene (TNF 50ng/ml) 7.7913358033 7.6731211392 0.1671807812 7.5549064751 NFkB reporter with desrabilized luciferase gene (Without TNF) 0.0437047052 0.0400742606 0.0041599958 191.4725569656 4.1717745721 0.0355349846 0.0409830919 average SD Negative control 0.0015676137 0.0001536241 NFkB reporter with stable luciferase gene 0.2884662746 0.0409162997 NFkB reporter with destabilized luciferase gene 0.0726666667 0.0005773503 Cells rapidly degrade the destabilized form of the luciferase protein and hence the background luciferase activity (noise level) is greatly reduced. fold change Negative control 1.0883896197 0.0972227921 NFkB reporter with stable luciferase gene 28.723291658 0.0192649866 NFkB reporter with destabilized luciferase gene 191.4725569656 4.1717745721 Due to lower background activity destabilized luciferase reporter gave better fold induction Sheet1 0.0001536241 0.0409162997 0.0005773503 Relative Luciferase Units Sheet2 0.0972227921 0.0192649866 4.1717745721 Fold Induction Sheet3 Chart2 1.0883896197 0.0972227921 28.723291658 0.0192649866 191.4725569656 4.1717745721 Sheet1 Luc/renilla Ratio average SD fold change NFkB reporter with stable luciferase gene (TNF 50ng/ml) 8.2896305439 8.2857009402 0.0055572989 28.723291658 0.0192649866 8.2817713365 NFkB reporter with stable luciferase gene (Without TNF) 0.3173984677 0.2884662746 0.0409162997 0.2595340816 Negative control (TNF 50ng/ml) 0.0018535114 0.0017061745 0.0001524078 1.0883896197 0.0972227921 0.0015491573 0.0017158547 Negative control 0.0016857211 0.0015676137 0.0001536241 0.0013939373 0.0016231827 NFkB reporter with desrabilized luciferase gene (TNF 50ng/ml) 7.7913358033 7.6731211392 0.1671807812 7.5549064751 NFkB reporter with desrabilized luciferase gene (Without TNF) 0.0437047052 0.0400742606 0.0041599958 191.4725569656 4.1717745721 0.0355349846 0.0409830919 average SD Negative control 0.0015676137 0.0001536241 NFkB reporter with stable luciferase gene 0.2884662746 0.0409162997 NFkB reporter with destabilized luciferase gene 0.0726666667 0.0005773503 Cells rapidly degrade the destabilized form of the luciferase protein and hence the background luciferase activity (noise level) is greatly reduced. fold change Negative control 1.0883896197 0.0972227921 NFkB reporter with stable luciferase gene 28.723291658 0.0192649866 NFkB reporter with destabilized luciferase gene 191.4725569656 4.1717745721 Due to lower background activity destabilized luciferase reporter gave better fold induction Sheet1 0.0001536241 0.0409162997 0.0005773503 Relative Luciferase Units Sheet2 0.0972227921 0.0192649866 4.1717745721 Fold Induction Sheet3 Chart2 1 0.1936817627 126.5298165138 11.2459655252 Sheet1 12 repeats of p53 binding sites average SD p53 reporter + DMSO 1 0.1936817627 p53 reporter + 1uM Doxorubicin 126.5298165138 11.2459655252 p53 reporter + 100uM Nutlin 63.630733945 8.4920671776 6 repeats of p53 binding site average SD p53 reporter + DMSO 1 0.0107580796 p53 reporter + 1uM Doxorubicin 2.4720496894 0.493818117 p53 reporter + 100uM Nutlin 3.1801242236 0.7360445001 12 repeats of p53 binding site gave best fold induction and hence incorporated in p53 reporter assay Sheet1 0.1936817627 11.2459655252 Relative Luciferase Units Sheet2 0.0107580796 0.493818117 Relative Luciferase Units Sheet3 Chart3 1 0.0107580796 2.4720496894 0.493818117 Sheet1 12 repeats of p53 binding sites average SD p53 reporter + DMSO 1 0.1936817627 p53 reporter + 1uM Doxorubicin 126.5298165138 11.2459655252 p53 reporter + 100uM Nutlin 63.630733945 8.4920671776 6 repeats of p53 binding site average SD p53 reporter + DMSO 1 0.0107580796 p53 reporter + 1uM Doxorubicin 2.4720496894 0.493818117 p53 reporter + 100uM Nutlin 3.1801242236 0.7360445001 12 repeats of p53 binding site gave best fold induction and hence incorporated in p53 reporter assay Sheet1 0.1936817627 11.2459655252 Relative Luciferase Units Sheet2 0.0107580796 0.493818117 Relative Luciferase Units Sheet3
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PowerPoint Presentation Contact: Anisha Kharkia; anisha.kharkia@qiagen.com Cignal Plasmid and Lenti Reporter Kits Sample to Insight 1 Cignal Plasmid and Lenti Reporter Kits Functional analysis of genes, biologics and small molecule compounds  The Cignal Reporter Assays are intended for molecular biology applications. These products are not intended for the diagnosis, prevention or treatment of a disease. Sample to Insight How you can use reporter assays in your research 3 Taking on challenges with reporter and lenti assays 1 How Cignal Reporter and Lenti Assays work 2 Agenda 3 Summary 4 Image reference: Apoptosis through death receptors. Pathway central, GeneGlobe. Sample to Insight 3 How you can use Cignal Reporter Assays in your research 3 Taking on challenges with reporter and lenti assays 1 How Cignal Reporter and Lenti Assays work 2 Agenda 4 Summary 4 Challenges associated a Solution: Cignal Reporter and Lenti Assay b Image reference: Apoptosis through death receptors. Pathway central, GeneGlobe. Sample to Insight 4 Research challenge: post-genomics Moving from a signaling flowchart perspective to a signaling network perspective DNA damage ATM/ATR p53 Cell cycle arrest/ apoptosis Basic signal transduction flowchart Sample to Insight 5 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 Sample to Insight Cell-based signaling studies: the challenges Frustration of analyzing one pathway at a time Time consuming Misleading Studies limited to cells that are easily transfected What about primary cells? What about stem cells? What about cell lines that are difficult to transfect? Sample to Insight 7 Solutions provided by Cignal Lenti Reporter Assays Engineered and formatted for superior performance Custom engineered transcriptional response elements (TRE) Use reporter genes with outstanding sensitivity and specificity Internal, positive and negative controls for reproducible results Luciferase and GFP formats are available Lentiviral particles are ready for transduction right out of the box Cignal Finder 10-Pathway Arrays Enable the rapid and reliable study of multiple signaling pathways Sample to Insight 8 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 to Insight Research challenge: constructing signaling networks Image reference: TNF pathway and interaction network. GeneGlobe Sample to Insight Research challenge: constructing signaling networks A B C D Signal transduction cascade (~40 proteins) Image reference: SMAD signaling network Sample to Insight 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) and destabilized, codon-optimized firefly luciferase Increases the signal to noise ratio, maximizes assay sensitivity and specificity Available in both luciferase and GFP formats Sample to Insight Agenda 13 How you can use Cignal Reporter Assays in your research 3 Taking on challenges with Cignal Reporter Assays 1 How Cignal Reporter and Lenti Assays work 2 Summary 4 Image reference: Apoptosis through death receptors. Pathway central, GeneGlobe. Sample to Insight 13 How you can use Cignal Reporter Assays in your research 3 Taking on challenges with Cignal Reporter Assays 1 How Cignal Reporter and Lenti Assays work 2 Agenda 14 Complete product portfolio a Dual-luciferase assays b Summary 4 High-performance luciferase a Pre-optimized TREs b Image reference: Apoptosis through death receptors. Pathway central, GeneGlobe. Sample to Insight 14 Two reporter modalities Dual-luciferase format Quantitative end-point luminescence assay Exceptional reproducibility, sensitivity and signal-to-noise ratio Average expression from a population of cells GFP format Dynamic live cell assay Single cell resolution Readout flexibility (flow cytometry, fluorescent microscopy, fluorometry) Sample to Insight 15 Flexible solution: multiple reporter formats Reporter protein is expressed when TF binds to TRE Reporter construct Pathway-targeted Transcriptional Regulatory Elements (TRE), which establish the pathway specificity of each reporter Sample to Insight 16 Reporter construct Tandem Repeats of TRE Pathway-targeted Transcriptional Regulatory Elements (TRE), which establish the pathway specificity of each reporter Upstream signaling events TF EGFP FL Flexible solution: multiple reporter formats Sample to Insight 17 Cignal Reporter and Lenti Reporter Assays: complete solution Rigorously optimized, functionally validated and ready-to-use reporters Positive control construct Negative control construct Internal control construct Reporter construct Pathway-targeted Transcriptional Regulatory Elements (TRE), which establish the pathway specificity of each reporter Sample to Insight 18 Positive control construct Negative control construct Internal control construct Multiple reporter formats: complete dual Luciferase system Reporter construct Pathway-targeted Transcriptional Regulatory Elements (TRE), which establish the pathway specificity of each reporter Sample to Insight 19 Easy-to- transfect cell lines Primary cells, stem cells and difficult-to-transfect cell lines End-point Assays Dynamic Live Cell Assays Dynamic Live Cell Assays Cignal Dual- Luciferace Reporter Assays Cignal GFP Reporter Assays Cignal Luc/ GFP Reporter Assays Cignal Lenti GFP / Luc Reporter Assays Single Pathway Assays 10-Pathway Arrays 45-Pathway Arrays Single Pathway Assays Single Pathway Assays Single Pathway Assays End-point Assays Cignal Lenti Luciferace Reporter Assays Cignal GFP Luciferase Reporter Assays 45-Pathway Arrays 10-Pathway Arrays Single Pathway Assays Single Pathway Assays Multiple options to suit your research needs: plasmid and lentiviral, luciferase and GFP reporters, single, 10-pathway, 45-pathway Cignal Reporter and Lenti Reporter Assays: product offering Sample to Insight Cignal Reporter Assays: Plasmid – 45 Pathways Pathway Transcription Factor Amino Acid Deprivation  ATF4/ATF3/ATF2  Androgen  Androgen Receptor  Antioxidant Response  Nrf2 & Nrf1  ATF6  ATF6  C/EBP C/EBP cAMP/PKA CREB Cell Cycle E2F/DP1 DNA Damage p53 EGR1  EGR1  ER Stress  CBF/NF-Y/YY1  Estrogen ER GATA GATA Glucocorticoid GR Heat Shock Response  HSF  Heavy Metal Stress  MTF1  Hedgehog Gli HNF4 HNF4 Hypoxia HIF-1 Interferon Regulation IRF-1 Type I Interferon STAT1/STAT2 Interferon Gamma STAT1/STAT1 KLF4 KLF4 Liver X LXR Pathway Transcription Factor MAPK/ERK Elk-1/SRF MAPK/JNK AP-1 MEF2  MEF2  c-myc Myc/Max Nanog Nanog NFκB NFκB  Notch RBP-Jk Oct4  Oct4  Pax6 Pax6 PI3K/AKT  FOXO  PKC/Ca++ NFAT PPAR  PPAR  Progesterone PR Retinoic Acid RAR Retinoid X RXR Sox2 Sox2 SP1  SP1  STAT3  STAT3  TGFβ SMAD2/3/4 VDRE  Vitamin D Receptor  Wnt TCF/LEF Xenobiotic AhR Sample to Insight Cignal Reporter Assays: Lentiviral Assays (35 Pathways) Pathway Transcription Factor Amino Acid Deprivation  ATF4/ATF3/ATF2  Androgen  Androgen Receptor  Antioxidant Response  Nrf2 & Nrf1  ATF6  ATF6  C/EBP C/EBP cAMP/PKA CREB Cell Cycle E2F/DP1 DNA Damage p53 EGR1  EGR1  ER Stress  CBF/NF-Y/YY1  Estrogen ER GATA GATA Glucocorticoid GR Heat Shock Response  HSF  Heavy Metal Stress  MTF1  Hedgehog Gli HNF4 HNF4 Hypoxia HIF-1 Interferon Regulation IRF-1 Type I Interferon STAT1/STAT2 Interferon Gamma STAT1/STAT1 KLF4 KLF4 Liver X LXR Pathway Transcription Factor MAPK/ERK Elk-1/SRF MAPK/JNK AP-1 MEF2  MEF2  c-myc Myc/Max Nanog Nanog NFκB NFκB  Notch RBP-Jk Oct4  Oct4  Pax6 Pax6 PI3K/AKT  FOXO  PKC/Ca++ NFAT PPAR  PPAR  Progesterone PR Retinoic Acid RAR Retinoid X RXR Sox2 Sox2 SP1  SP1  STAT3  STAT3  TGFβ SMAD2/3/4 VDRE  Vitamin D Receptor  Wnt TCF/LEF Xenobiotic AhR Sample to Insight 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 Sample to Insight Using Cignal Lenti Reporter Assay What you need: Cignal Lenti Reporter or 10-Pathway Array SureENTRY™ can enhance transduction efficiency in most cell types Test biological siRNA (Flexitube siRNA) shRNA (SureSilencing shRNA) expression vectors (Qiagenes) protein/peptide small molecule Luciferase Assay Kit (Promega) and luminometer or GFP detection system Sample to Insight 24 Cignal Reporter & Lenti Reporter Assays Reporter construct Normalization construct Pathway-targeted transcriptional regulatory elements (45) Constitutive transcriptional regulatory element (1) The Transcriptional Regulatory Elements (TREs) establishe the pathway specificity of each reporter! Key advantages, assay design, dual-luciferase Sample to Insight Cignal Reporter and Lenti 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 Changes due to the designed treatments of cells RNA interference Overexpression Recombinant protein/peptide/growth factor Small molecule Dual-luciferase assay design corrects for these unwanted sources of variability. Sample to Insight Dual-luciferase assays: NFkB Reporter Assay CV=0.09 CV=0.07 Single-luciferase assays: NFkB Reporter Assay CV=0.69 CV=0.70 NFkB Reporter + NFkB siRNA NFkB Reporter + Neg. control siRNA NFkB Reporter + NFkB siRNA NFkB Reporter + Neg. control siRNA Dual-luciferase format minimizes variability Relative Luciferase Units Luminescence (Relative Light Units) Sample to Insight Modified luciferase: background NFkB Reporter signal (no treatment post-transfection) Modified luciferase: NFkB Reporter signal following TNF induction WHY? Negative control NFkB Reporter: Stable Luc NFkB Reporter: Destabilized Luc Negative control NFkB Reporter: Stable Luc NFkB Reporter: Destabilized Luc Advantage: genetic engineered luciferase High signal-to-noise ratio offers higher sensitivity Relative Luciferase Units Fold Induction Sample to Insight Dual-luciferase assays utilizing Cignal p53 TRE p53 Reporter + DMSO p53 Reporter + 1µM Doxorubicin Dual-luciferase assays utilizing common p53 TRE p53 Reporter + DMSO p53 Reporter + 1µM Doxorubicin 125-Fold 2.5-Fold TRE maximizes luciferase signal Relative Luciferase Units Relative Luciferase Units Sample to Insight Cignal Reporter and Lenti Reporter optimization process Improved sensitivity and biological relevance: Experimentally optimized response elements : Sequence of transcription response element (TRE) Number of TREs Intervening sequence between TRE Engineered reporter genes: Destabilized firefly luciferase (Maximize signal-to-noise ratio) Codon optimization for mammalian expression (Maximize expression) Removal of hidden transcription factor binding sites (Reduce background) Maximize specificity and sensitivity Save time, money and labor: use Cignal Reporters and Lenti Reporters Sample to Insight 30 Cignal Reporter Assays Pathway Transcription Factor Amino Acid Deprivation  ATF4/ATF3/ATF2  Androgen  Androgen Receptor  Antioxidant Response  Nrf2 & Nrf1  ATF6  ATF6  C/EBP C/EBP cAMP/PKA CREB Cell Cycle E2F/DP1 DNA Damage p53 EGR1  EGR1  ER Stress  CBF/NF-Y/YY1  Estrogen ER GATA GATA Glucocorticoid GR Heat Shock Response  HSF  Heavy Metal Stress  MTF1  Hedgehog Gli HNF4 HNF4 Hypoxia HIF-1 Interferon Regulation IRF-1 Type I Interferon STAT1/STAT2 Interferon Gamma STAT1/STAT1 KLF4 KLF4 Liver X LXR Pathway Transcription Factor MAPK/ERK Elk-1/SRF MAPK/JNK AP-1 MEF2  MEF2  c-myc Myc/Max Nanog Nanog NFκB NFκB  Notch RBP-Jk Oct4  Oct4  Pax6 Pax6 PI3K/AKT  FOXO  PKC/Ca++ NFAT PPAR  PPAR  Progesterone PR Retinoic Acid RAR Retinoid X RXR Sox2 Sox2 SP1  SP1  STAT3  STAT3  TGFβ SMAD2/3/4 VDRE  Vitamin D Receptor  Wnt TCF/LEF Xenobiotic AhR DNA-vector GFP – 16 pathways Sample to Insight Cignal Lenti Reporter Assays Pathway Transcription Factor Amino Acid Deprivation  ATF4/ATF3/ATF2  Androgen  Androgen Receptor  Antioxidant Response  Nrf2 & Nrf1  ATF6  ATF6  C/EBP C/EBP cAMP/PKA CREB Cell Cycle E2F/DP1 DNA Damage p53 EGR1  EGR1  ER Stress  CBF/NF-Y/YY1  Estrogen ER GATA GATA Glucocorticoid GR Heat Shock Response  HSF  Heavy Metal Stress  MTF1  Hedgehog Gli HNF4 HNF4 Hypoxia HIF-1 Interferon Regulation IRF-1 Type I Interferon STAT1/STAT2 Interferon Gamma STAT1/STAT1 KLF4 KLF4 Liver X LXR Pathway Transcription Factor MAPK/ERK Elk-1/SRF MAPK/JNK AP-1 MEF2  MEF2  c-myc Myc/Max Nanog Nanog NFκB NFκB  Notch RBP-Jk Oct4  Oct4  Pax6 Pax6 PI3K/AKT  FOXO  PKC/Ca++ NFAT PPAR  PPAR  Progesterone PR Retinoic Acid RAR Retinoid X RXR Sox2 Sox2 SP1  SP1  STAT3  STAT3  TGFβ SMAD2/3/4 VDRE  Vitamin D Receptor  Wnt TCF/LEF Xenobiotic AhR Lenti-GFP Reporter – 6 pathways Sample to Insight Treatment of Cignal SRE (GFP) transfectants with 10% serum and 10 ng/ml PMA activates the MAPK/ERK signaling pathway. Fluorometry Fluorescence microscopy Cignal Reporter Assays GFP system application: small molecule studies Sample to Insight Agenda 34 How you can use Cignal Reporter Assays in your research 3 Taking on challenges with Cignal Reporter Assays 1 How Cignal Reporter and Lenti Assays work 2 Summary 4 Image reference: Apoptosis through death receptors. Pathway central, GeneGlobe. Sample to Insight 34 Cignal Lenti Reporters: primary cells PKC/Ca++ signaling pathway studies in primary arteriole smooth muscle cells Cignal Lenti Reporter system makes this study possible Relative Luciferase Units Sample to Insight Cignal Lenti Reporters: challenging cell lines NFkB signaling pathway studies in D1 Murine T-cell Leukemia cell line Cignal Lenti Reporter system makes this study possible Sample to Insight Cignal Lenti Reporters: challenging cell lines Cignal Lenti SRE Reporter (GFP) measures MAPK/ERK pathway signaling activity in African green monkey CV1 fibroblasts Cignal Lenti Reporter system makes this study possible Sample to Insight Cignal Lenti Reporters: sensor cell lines Generation of an NFkB signaling pathway stable sensor cell line Cignal Lenti Reporters enable rapid pathway sensor cell line generation P1 P5 P10 P15 Relative Luciferase Units Sample to Insight 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”? Cignal Reporter Assays: application in multiple research areas Sample to Insight Cignal Reporter Assays: application – RNA interference Relative Luciferase Units Functional genomics What’s the phenotype of my gene? Does my gene of interest regulate signaling pathway “X”? Sample to Insight Cignal Reporter Assays: application Functional proteomics What’s the phenotype of my protein/peptide? Does my protein, peptide, or growth factor of interest regulate signaling pathway “X”? Sample to Insight Cignal Reporter Assays: application – small molecule studies Relative Luciferase Units Drug screening What is the mechanism of action of my small molecule drug candidates? Do my drug candidates of interest regulate signaling pathway “X”? Sample to Insight Situation: I have a biological molecule that knocks down p53 expression Questions: What is impact of p53 gene expression knock down on the p53 signaling pathway? What is the impact of p53 knock down on other cancer-relevant signaling pathways? Research challenge: pathway interaction study Sample to Insight Wnt signaling NFkB signaling E2F signaling MAPK/ERK signaling Myc signaling MAPK/JNK signaling TGFβ signaling Notch signaling ? ? ? ? ? ? ? ? ? Hypoxia signaling p53 signaling Research challenge: pathway interactions Image reference: p53signaling. Pathway central, GeneGlobe. Sample to Insight Approach: Carry out a p53 RNA interference experiment with multiple cancer-relevant Cignal Reporter Assays Cignal Reporter Assays: application – study pathway interaction Sample to Insight Cignal Finder Arrays: cancer array design Pathway Cell Cycle DNA Damage Hypoxia MAPK/ERK MAPK/JNK c-Myc NFkB Notch TGFβ Wnt Transcription Factor E2F/DP1 p53 HIF Elk-1/SRF AP1 Myc/Max NFkB RBP-Jk SMAD2/SMAD3/SMAD4 TCF/LEF Sample to Insight 46 Cignal Finder Array: how does it work? Sample to Insight Cignal Finder Cancer Array: p53 RNAi study design p53 Cignal Reporter RBP-Jk Cignal Reporter TCF/LEF Cignal Reporter E2F Cignal Reporter AP1 Cignal Reporter SRE Cignal Reporter SMAD Cignal Reporter 8. NFkB Cignal Reporter 9. Myc Cignal Reporter 10. HIF Cignal Reporter 11. Cignal Negative control 12. Cignal Positive control p53 siRNA Neg. control siRNA Sample to Insight Impact of p53 siRNA treatment Cignal Finder Cancer Array: p53 RNAi study design – results Fold Change in Pathway Activation Sample to Insight MAPK/ERK signaling Notch signaling Hypoxia signaling p53 signaling Cignal Finder Cancer Array: p53 RNAi study design – next steps Image reference: p53signaling. Pathway central, GeneGlobe. Sample to Insight Cignal Lenti Reporters: biosafety VSV-G pseudotyped lentiviral particles Deletion in U3 portion of 3’LTR results in self-inactivation (SIN), following transduction of target cell No structural or replication genes are expressed in transduced cells, making Cignal lentiviral vectors replication-defective No virulence genes (vpr, vif, vpu, nef) are present in the Cignal Lenti Reporters Experiments should be performed under Biosafety Level 2 (BSL-2) conditions See our Cignal Lenti Reporter web pages for useful links Sample to Insight Tips for a successful transduction Avoid freeze thaws Result in activity loss MOI dilution series Recommended range 5–50 Transduction reagent Sure ENTRY usually in the range of 4–8 μg/ml MOIs >50 should be split Sequential 4-hour transductions Sample to Insight Tips for making stable cell lines Kill curve for Puromycin Don’t guess 500–10,000 ng/ml MOI for stables Perform a transient study to find appropriate MOI Freeze cells down Renewable reagent Sample to Insight Agenda 54 How you can use Cignal Reporter Assays in your research 3 Taking on challenges with Cignal Reporter Assays 1 How Cignal Reporter and Lenti Assays work 2 Summary 4 Image reference: Apoptosis through death receptors. Pathway central, GeneGlobe. Sample to Insight 54 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 Final benefit to researchers: Ready-to-use, validated reporters Don’t have to produce or amplify lentivirus in your laboratory Quick generation of reporter cell lines Sample to Insight 55 Summary: Cignal Reporter Assays system Breadth of pathways and research application 45 pathway-focused reporter assays 6 application-specific Cignal Finder 10-Pathway Arrays 1 Cignal 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 end-point assays GFP dynamic live cell assays with single cell resolution Sample to Insight Cignal Reporter and Lenti Reporter Assays 57 Questions? Contact technical support 9 AM – 6 PM EST Telephone: 800-362-7737 Or Contact Anisha Kharkia Global Product Manager Email: Anisha.Kharkia@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. Sample to Insight 57 Thank you Sample to Insight Chart1 5727326 10223380 2003220 15737362 23907364 3733934 Sheet1 Luciferase Renilla ratio Average SD CV NFkB reporter + NFkB1 siRNA 5727326 6051066 0.946 0.8753333333 0.0783985544 0.09 10223380 11495530 0.889 2003220 2531686 0.791 NFkB reporter + negative control siRNA 15737362 6968666 2.258 2.1156666667 0.1477712196 0.07 23907364 12176456 1.963 3733934 1756248 2.126 NFkB reporter + NFkBIA shRNA 6573910 3317632 1.982 2.3169052671 0.2982141376 0.13 86515384 35828692 2.415 36551702 14313144 2.5537158014 NFkB reporter + Rel A shRNA 2223454 4943094 0.45 0.5517868727 0.0947267092 0.17 6903218 12143928 0.568 3329888 5224496 0.6373606181 NFkB reporter + negative control shRNA 47583702 47919378 0.993 1.1472143585 0.1922742808 0.17 31502328 29003080 1.086 20282686 14884812 1.3626430754 SD CV Luciferase Luciferase Luciferase Avg. Triplicate 4116117 0.69 NFkB reporter + NFkB1 siRNA 5727326 10223380 2003220 5984642 10147237 0.7 NFkB reporter + negative control siRNA 15737362 23907364 3733934 14459553 40384974 0.93 NFkB reporter + NFkBIA shRNA 6573910 86515384 36551702 43213665 2445849 0.59 NFkB reporter + Rel A shRNA 2223454 6903218 3329888 4152187 13722466 0.41 NFkB reporter + negative control shRNA 47583702 31502328 20282686 33122905 Luciferase/Renilla ratio NFkB reporter + NFkB1 siRNA 0.946 0.889 0.791 NFkB reporter + negative control siRNA 2.258 1.963 2.126 NFkB reporter + NFkBIA shRNA 1.982 2.415 2.5537158014 NFkB reporter + Rel A shRNA 0.45 0.568 0.6373606181 NFkB reporter + negative control shRNA 0.993 1.086 1.3626430754 Use of dual luciferase, which is inbuild in cignal reporter assay, reduce experimental variability and provide more realiable results Sheet1 Luminescence (Relative Light Units) Sheet2 Relative Luciferase Units Sheet3 Chart2 0.946 0.889 0.791 2.258 1.963 2.126 Sheet1 Luciferase Renilla ratio Average SD CV NFkB reporter + NFkB1 siRNA 5727326 6051066 0.946 0.8753333333 0.0783985544 0.09 10223380 11495530 0.889 2003220 2531686 0.791 NFkB reporter + negative control siRNA 15737362 6968666 2.258 2.1156666667 0.1477712196 0.07 23907364 12176456 1.963 3733934 1756248 2.126 NFkB reporter + NFkBIA shRNA 6573910 3317632 1.982 2.3169052671 0.2982141376 0.13 86515384 35828692 2.415 36551702 14313144 2.5537158014 NFkB reporter + Rel A shRNA 2223454 4943094 0.45 0.5517868727 0.0947267092 0.17 6903218 12143928 0.568 3329888 5224496 0.6373606181 NFkB reporter + negative control shRNA 47583702 47919378 0.993 1.1472143585 0.1922742808 0.17 31502328 29003080 1.086 20282686 14884812 1.3626430754 SD CV Luciferase Luciferase Luciferase Avg. Triplicate 4116117 0.69 NFkB reporter + NFkB1 siRNA 5727326 10223380 2003220 5984642 10147237 0.7 NFkB reporter + negative control siRNA 15737362 23907364 3733934 14459553 40384974 0.93 NFkB reporter + NFkBIA shRNA 6573910 86515384 36551702 43213665 2445849 0.59 NFkB reporter + Rel A shRNA 2223454 6903218 3329888 4152187 13722466 0.41 NFkB reporter + negative control shRNA 47583702 31502328 20282686 33122905 Luciferase/Renilla ratio NFkB reporter + NFkB1 siRNA 0.946 0.889 0.791 NFkB reporter + negative control siRNA 2.258 1.963 2.126 NFkB reporter + NFkBIA shRNA 1.982 2.415 2.5537158014 NFkB reporter + Rel A shRNA 0.45 0.568 0.6373606181 NFkB reporter + negative control shRNA 0.993 1.086 1.3626430754 Use of dual luciferase, which is inbuild in cignal reporter assay, reduce experimental variability and provide more realiable results Sheet1 Luminescence (Relative Light Units) Sheet2 Relative Luciferase Units Sheet3 Chart1 0.0015676137 0.0001536241 0.2884662746 0.0409162997 0.0726666667 0.0005773503 Sheet1 Luc/renilla Ratio average SD fold change NFkB reporter with stable luciferase gene (TNF 50ng/ml) 8.2896305439 8.2857009402 0.0055572989 28.723291658 0.0192649866 8.2817713365 NFkB reporter with stable luciferase gene (Without TNF) 0.3173984677 0.2884662746 0.0409162997 0.2595340816 Negative control (TNF 50ng/ml) 0.0018535114 0.0017061745 0.0001524078 1.0883896197 0.0972227921 0.0015491573 0.0017158547 Negative control 0.0016857211 0.0015676137 0.0001536241 0.0013939373 0.0016231827 NFkB reporter with desrabilized luciferase gene (TNF 50ng/ml) 7.7913358033 7.6731211392 0.1671807812 7.5549064751 NFkB reporter with desrabilized luciferase gene (Without TNF) 0.0437047052 0.0400742606 0.0041599958 191.4725569656 4.1717745721 0.0355349846 0.0409830919 average SD Negative control 0.0015676137 0.0001536241 NFkB reporter with stable luciferase gene 0.2884662746 0.0409162997 NFkB reporter with destabilized luciferase gene 0.0726666667 0.0005773503 Cells rapidly degrade the destabilized form of the luciferase protein and hence the background luciferase activity (noise level) is greatly reduced. fold change Negative control 1.0883896197 0.0972227921 NFkB reporter with stable luciferase gene 28.723291658 0.0192649866 NFkB reporter with destabilized luciferase gene 191.4725569656 4.1717745721 Due to lower background activity destabilized luciferase reporter gave better fold induction Sheet1 0.0001536241 0.0409162997 0.0005773503 Relative Luciferase Units Sheet2 0.0972227921 0.0192649866 4.1717745721 Fold Induction Sheet3 Chart2 1.0883896197 0.0972227921 28.723291658 0.0192649866 191.4725569656 4.1717745721 Sheet1 Luc/renilla Ratio average SD fold change NFkB reporter with stable luciferase gene (TNF 50ng/ml) 8.2896305439 8.2857009402 0.0055572989 28.723291658 0.0192649866 8.2817713365 NFkB reporter with stable luciferase gene (Without TNF) 0.3173984677 0.2884662746 0.0409162997 0.2595340816 Negative control (TNF 50ng/ml) 0.0018535114 0.0017061745 0.0001524078 1.0883896197 0.0972227921 0.0015491573 0.0017158547 Negative control 0.0016857211 0.0015676137 0.0001536241 0.0013939373 0.0016231827 NFkB reporter with desrabilized luciferase gene (TNF 50ng/ml) 7.7913358033 7.6731211392 0.1671807812 7.5549064751 NFkB reporter with desrabilized luciferase gene (Without TNF) 0.0437047052 0.0400742606 0.0041599958 191.4725569656 4.1717745721 0.0355349846 0.0409830919 average SD Negative control 0.0015676137 0.0001536241 NFkB reporter with stable luciferase gene 0.2884662746 0.0409162997 NFkB reporter with destabilized luciferase gene 0.0726666667 0.0005773503 Cells rapidly degrade the destabilized form of the luciferase protein and hence the background luciferase activity (noise level) is greatly reduced. fold change Negative control 1.0883896197 0.0972227921 NFkB reporter with stable luciferase gene 28.723291658 0.0192649866 NFkB reporter with destabilized luciferase gene 191.4725569656 4.1717745721 Due to lower background activity destabilized luciferase reporter gave better fold induction Sheet1 0.0001536241 0.0409162997 0.0005773503 Relative Luciferase Units Sheet2 0.0972227921 0.0192649866 4.1717745721 Fold Induction Sheet3 Chart2 1 0.1936817627 126.5298165138 11.2459655252 Sheet1 12 repeats of p53 binding sites average SD p53 reporter + DMSO 1 0.1936817627 p53 reporter + 1uM Doxorubicin 126.5298165138 11.2459655252 p53 reporter + 100uM Nutlin 63.630733945 8.4920671776 6 repeats of p53 binding site average SD p53 reporter + DMSO 1 0.0107580796 p53 reporter + 1uM Doxorubicin 2.4720496894 0.493818117 p53 reporter + 100uM Nutlin 3.1801242236 0.7360445001 12 repeats of p53 binding site gave best fold induction and hence incorporated in p53 reporter assay Sheet1 0.1936817627 11.2459655252 Relative Luciferase Units Sheet2 0.0107580796 0.493818117 Relative Luciferase Units Sheet3 Chart3 1 0.0107580796 2.4720496894 0.493818117 Sheet1 12 repeats of p53 binding sites average SD p53 reporter + DMSO 1 0.1936817627 p53 reporter + 1uM Doxorubicin 126.5298165138 11.2459655252 p53 reporter + 100uM Nutlin 63.630733945 8.4920671776 6 repeats of p53 binding site average SD p53 reporter + DMSO 1 0.0107580796 p53 reporter + 1uM Doxorubicin 2.4720496894 0.493818117 p53 reporter + 100uM Nutlin 3.1801242236 0.7360445001 12 repeats of p53 binding site gave best fold induction and hence incorporated in p53 reporter assay Sheet1 0.1936817627 11.2459655252 Relative Luciferase Units Sheet2 0.0107580796 0.493818117 Relative Luciferase Units Sheet3
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