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Sample & Assay Technologies - 1 - Cignal Reporter Assays (45 Pathways) PathwayTranscription Factor Amino Acid DeprivationAmino Acid Deprivation ATF4/ATF3/ATF2.

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Presentation on theme: "Sample & Assay Technologies - 1 - Cignal Reporter Assays (45 Pathways) PathwayTranscription Factor Amino Acid DeprivationAmino Acid Deprivation ATF4/ATF3/ATF2."— Presentation transcript:

1 Sample & Assay Technologies Cignal Reporter Assays (45 Pathways) PathwayTranscription Factor Amino Acid DeprivationAmino Acid Deprivation ATF4/ATF3/ATF2 ATF4/ATF3/ATF2 AndrogenAndrogen Androgen Receptor Androgen Receptor Antioxidant ResponseAntioxidant Response Nrf2 & Nrf1 Nrf2 & Nrf1 ATF6ATF6 ATF6 ATF6 C/EBP cAMP/PKAcAMP/PKA CREB Cell Cycle E2F/DP1CREB Cell CycleE2F/DP1 DNA DamageDNA Damage p53p53 EGR1EGR1 EGR1 EGR1 ER StressER Stress CBF/NF-Y/YY1 CBF/NF-Y/YY1 EstrogenEstrogen ERER GATA GlucocorticoidGlucocorticoid GRGR Heat Shock ResponseHeat Shock Response HSF HSF Heavy Metal StressHeavy Metal Stress MTF1 MTF1 HedgehogGLI HNF4 HypoxiaHypoxia HIF-1HIF-1 Interferon RegulationIRF-1 Type I InterferonType I Interferon STAT1/STAT2STAT1/STAT2 Interferon GammaInterferon Gamma STAT1/STAT1STAT1/STAT1 KLF4 Liver XLXR PathwayTranscription Factor MAPK/ERKMAPK/ERK Elk-1/SRFElk-1/SRF MAPK/JNKMAPK/JNK AP-1AP-1 MEF2MEF2 MEF2 MEF2 c-mycc-myc Myc/MaxMyc/Max Nanog NFκB Notch RBP-Jk Oct4Oct4 Oct4 Oct4 Pax6 PI3K/AKTPI3K/AKT FOXO FOXO PKC/Ca++ NFAT PPARPPAR PPAR PPAR ProgesteronePR Retinoic Acid RAR Retinoid XRXR Sox2 SP1SP1 SP1 SP1 STAT3STAT3 STAT3 STAT3 TGFβTGFβ SMAD2/3/4SMAD2/3/4 Vitamin DVitamin D Receptor Wnt TCF/LEF XenobioticAhR

2 Sample & Assay Technologies Cignal™ Reporter Assays Samuel J. Rulli, Jr., Ph.D. qPCR and Pathway Signaling Application Scientist Cell-Based Assays for Rapidly Analyzing Pathway Signaling Activity

3 Sample & Assay Technologies Topics to be Covered Utility of the Cignal Reporter Assays Challenges Associated with Cell-Based Assays Solutions Cignal Reporter Assays Provide. How Cignal Reporter Assays Work Cignal Pathway Reporters Product Family Dual-Luciferase Assays Luciferase Engineered for High Performance Optimized Transcriptional Regulatory Elements (TREs). Application Study: p53 Pathways

4 Sample & Assay Technologies Post-Genomics Research Challenge: Moving from a Signaling Flowchart Perspective to a Signaling Network Perspective. DNA damage ATM/ATR p53 Cell cycle arrest/Apoptosis Basic Signal Transduction Flowchart

5 Sample & Assay Technologies Post-Genomics Research Challenge: Signal Transduction Cascade A B C D (~40 proteins)

6 Sample & Assay Technologies Cell-based Assay Research Challenges. Variability within experiments Well-to-well and plate-to-plate reproducibility are notoriously challenging with cell-based assays.. Variability between experiments Reliable results often depend upon the “status” of the cells, and who in the lab is conducting the experiments.. Assay Performance Sensitivity, specificity, signal to noise ratio, and simplicity are of utmost importance.

7 Sample & Assay Technologies What Cignal Reporter Assays Provide Functionally validated dual-reporter formulation Minimizes experimental variability, increasing the biological relevance Ready-to-use reporters and controls, in transfection ready format: Enables simple and rapid analysis of the regulation of 45 signal transduction pathways Custom-engineered transcriptional regulatory elements (TREs) and destabilized, codon-optimized firefly luciferase Increases the signal to noise ratio and maximizes assay sensitivity and specificity

8 Sample & Assay Technologies Topics to be Covered Utility of the Cignal Reporter Assays Challenges Associated with Cell-Based Assays Solutions Cignal Reporter Assays Provide. How Cignal Reporter Assays Work Cignal Pathway Reporters Product Family Dual-Luciferase Assays Luciferase Engineered for High Performance Optimized Transcriptional Regulatory Elements (TREs). Application Study: p53 Pathways

9 Sample & Assay Technologies Cignal Pathway Reporter System Products Easy to transfect cell lines Primary cells, stem cells, and difficult to transfect cell lines Endpoint Assays Endpoint Assays 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 Pathway Assays Single Pathway Assays Single Pathway Assays Single Pathway Assays 10-Pathway Arrays 10-Pathway Arrays

10 Sample & Assay Technologies Cignal Reporter Assays (45 Pathways) PathwayTranscription Factor Amino Acid DeprivationAmino Acid Deprivation ATF4/ATF3/ATF2 ATF4/ATF3/ATF2 AndrogenAndrogen Androgen Receptor Androgen Receptor Antioxidant ResponseAntioxidant Response Nrf2 & Nrf1 Nrf2 & Nrf1 ATF6ATF6 ATF6 ATF6 C/EBP cAMP/PKAcAMP/PKA CREB Cell Cycle E2F/DP1CREB Cell CycleE2F/DP1 DNA DamageDNA Damage p53p53 EGR1EGR1 EGR1 EGR1 ER StressER Stress CBF/NF-Y/YY1 CBF/NF-Y/YY1 EstrogenEstrogen ERER GATA GlucocorticoidGlucocorticoid GRGR Heat Shock ResponseHeat Shock Response HSF HSF Heavy Metal StressHeavy Metal Stress MTF1 MTF1 HedgehogGLI HNF4 HypoxiaHypoxia HIF-1HIF-1 Interferon RegulationIRF-1 Type I InterferonType I Interferon STAT1/STAT2STAT1/STAT2 Interferon GammaInterferon Gamma STAT1/STAT1STAT1/STAT1 KLF4 Liver XLXR PathwayTranscription Factor MAPK/ERKMAPK/ERK Elk-1/SRFElk-1/SRF MAPK/JNKMAPK/JNK AP-1AP-1 MEF2MEF2 MEF2 MEF2 c-mycc-myc Myc/MaxMyc/Max Nanog NFκB Notch RBP-Jk Oct4Oct4 Oct4 Oct4 Pax6 PI3K/AKTPI3K/AKT FOXO FOXO PKC/Ca++ NFAT PPARPPAR PPAR PPAR ProgesteronePR Retinoic Acid RAR Retinoid XRXR Sox2 SP1SP1 SP1 SP1 STAT3STAT3 STAT3 STAT3 TGFβTGFβ SMAD2/3/4SMAD2/3/4 Vitamin DVitamin D Receptor Wnt TCF/LEF XenobioticAhR

11 Sample & Assay Technologies Cignal Lenti Assays (35 Pathways) PathwayTranscription Factor Amino Acid Deprivation ATF4/ATF3/ATF2 Androgen Androgen Receptor Antioxidant Response Nrf2 & Nrf1 ATF6 ATF6 C/EBP cAMP/PKA CREB Cell Cycle E2F/DP1 DNA Damage p53 EGR1 EGR1 ER Stress CBF/NF-Y/YY1 Estrogen ER GATA Glucocorticoid GR Heat Shock Response HSF Heavy Metal Stress MTF1 HedgehogGliHNF4 Hypoxia HIF-1 Interferon RegulationIRF-1 Type I Interferon STAT1/STAT2 Interferon Gamma STAT1/STAT1KLF4 Liver XLXR PathwayTranscription Factor MAPK/ERK Elk-1/SRF MAPK/JNK AP-1 MEF2 MEF2 c-myc Myc/MaxNanogNFκB Notch RBP-Jk Oct4 Oct4 Pax6 PI3K/AKT FOXO PKC/Ca++ NFAT PPAR PPAR ProgesteronePR Retinoic Acid RAR Retinoid XRXRSox2 SP1 SP1 STAT3 STAT3 TGFβ SMAD2/3/4 VDRE Vitamin D Receptor Wnt TCF/LEF XenobioticAhR

12 Sample & Assay Technologies REPORTER DESIGN: Dual-Luciferase Assays Reporter Construct Firefly luciferase TATA box TRERenilla luciferase TATA box CMV Normalization Construct Pathway-targeted transcriptional regulatory elements (45) Constitutive transcriptional regulatory element (1) The Transcriptional Regulatory Elements (TREs) are what establish the pathway specificity of each reporter!

13 Sample & Assay Technologies Using Cignal Reporters: 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

14 Sample & Assay Technologies REPORTER DESIGN: Dual-Luciferase Assays Reporter Construct Firefly luciferase TATA box TRERenilla luciferase TATA box CMV Normalization Construct Pathway-targeted transcriptional regulatory elements (45) Constitutive transcriptional regulatory element (1) The Transcriptional Regulatory Elements (TREs) are what establish the pathway specificity of each reporter!

15 Sample & Assay Technologies Dual Luciferase Assays: Why Is It Important? 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.

16 Sample & Assay Technologies Dual Luciferase Format Minimizes Variability 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

17 Sample & Assay Technologies REPORTER DESIGN: Dual-Luciferase Assays Reporter Construct Firefly luciferase TATA box TRERenilla luciferase TATA box CMV Normalization Construct Mammalian codon-optimized AND Destabilized firefly luciferase The Transcriptional Regulatory Elements (TREs) are what establish the pathway specificity of each reporter!

18 Sample & Assay Technologies Engineered Luciferase and the Signal:Noise Ratio 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

19 Sample & Assay Technologies REPORTER DESIGN: Dual-Luciferase Assays Reporter Construct Firefly luciferase TATA box TRERenilla luciferase TATA box CMV Normalization Construct Pathway-targeted transcriptional regulatory elements (45) The Transcriptional Regulatory Elements (TREs) are what establish the pathway specificity of each reporter!

20 Sample & Assay Technologies Cignal TRE Maximize Luciferase Signal 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

21 Sample & Assay Technologies Cignal Reporter Assays: How It Works

22 Sample & Assay Technologies Cignal Reporter Assays (GFP) PathwayTranscription Factor Amino Acid Deprivation ATF4/ATF3/ATF2 Androgen Androgen Receptor Antioxidant Response Nrf2 & Nrf1 ATF6 ATF6 C/EBP cAMP/PKA CREB Cell Cycle E2F/DP1 DNA Damage p53 EGR1 EGR1 ER Stress CBF/NF-Y/YY1 Estrogen ER GATA Glucocorticoid GR Heat Shock Response HSF Heavy Metal Stress MTF1 HedgehogGliHNF4 Hypoxia HIF-1 Interferon RegulationIRF-1 Type I Interferon STAT1/STAT2 Interferon Gamma STAT1/STAT1KLF4 Liver XLXR PathwayTranscription Factor MAPK/ERK Elk-1/SRF MAPK/JNK AP-1 MEF2 MEF2 c-myc Myc/MaxNanog NFκB NFκB Notch RBP-Jk Oct4 Oct4 Pax6 PI3K/AKT FOXO PKC/Ca++ NFAT PPAR PPAR ProgesteronePR Retinoic Acid RAR Retinoid XRXRSox2 SP1 SP1 STAT3 STAT3 TGFβ SMAD2/3/4 VDRE Vitamin D Receptor Wnt TCF/LEF XenobioticAhR

23 Sample & Assay Technologies GFP-based Reporter: Small Molecule Studies Treatment of Cignal SRE (GFP) transfectants with 10% serum and 10 ng/ml PMA activates the MAPK/ERK signaling pathway. Fluorometry Fluorescent Microscopy

24 Sample & Assay Technologies Cignal Reporter Assays: Two Modalities Dual-luciferase Format - Quantitative endpoint 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)

25 Sample & Assay Technologies Topics to be Covered Utility of the Cignal Reporter Assays Challenges Associated with Cell-Based Assays Solutions Cignal Reporter Assays Provide. How Cignal Reporter Assays Work Cignal Pathway Reporters Product Family Dual-Luciferase Assays Luciferase Engineered for High Performance Optimized Transcriptional Regulatory Elements (TREs). Application Study: p53 Pathways

26 Sample & Assay Technologies Application Study Question: What is impact of p53 gene expression knock down on the p53 signaling pathway AND other cancer-relevant signaling pathways? p53 Pathway Interaction Study

27 Sample & Assay Technologies Post-Genomic Challenges: Pathway Interactions Wnt SignalingNFkB Signaling E2F Signaling MAPK/ERK Signaling Myc Signaling MAPK/JNK Signaling TGFβ Signaling Notch Signaling ? ? ? ? ? ? ? ? ? Hypoxia Signaling p53 Signaling

28 Sample & Assay Technologies Constructing Signaling Networks

29 Sample & Assay Technologies Application Study Question: What is impact of p53 gene expression knock down on the p53 signaling pathway AND other cancer-relevant signaling pathways? Approach: Carry out a p53 RNA interference experiment with multiple cancer-relevant Cignal Reporter Assays. p53 Pathway Interaction Study

30 Sample & Assay Technologies Cignal Finder Cancer Array : p53 RNAi Study 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 10. HIF Cignal Reporter 11. Cignal Negative Control 12. Cignal Positive Control p53 siRNA Neg. control siRNA

31 Sample & Assay Technologies Results: Cancer Array Application Study Impact of p53 siRNA Treatment

32 Sample & Assay Technologies Post-Genomics Challenge: Pathway Interactions MAPK/ERK Signaling Notch Signaling Hypoxia Signaling p53 Signaling

33 Sample & Assay Technologies Summary: Cignal Reporter Assay System. Breadth of Pathways and Research Applications 45 Pathway-Focused Reporter Assays available individually or as biologically focused panels.. Performance Exceptional Sensitivity, Specificity, Signal-to-Noise Ratio, and Reproducibility. Ready-to-use, validated, preformatted transient assays.. Multiple Reporters: Dual-luciferase quantitative endpoint assays. GFP dynamic live cell assays with single cell resolution.


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