cignal reporter assays webinar 2013
TRANSCRIPT
Sample & Assay Technologies- 1 -For Internal Use Only
Cignal™ Reporter Assays
Krishnan Allampallam, Ph.D.,MBAPCR Array & Pathway Signaling
Functional Analysis of GenesBiologics and Small Molecule Compounds
The Cignal Reporter Assays are intended for molecular biology applications. This product is not intended for the diagnosis, prevention or treatment of a disease.
Sample & Assay Technologies- 2 -For Internal Use Only
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
Sample & Assay Technologies- 3 -For Internal Use Only
Research challengePost-Genomics
Moving from a Signaling Flowchart Perspectiveto a Signaling Network Perspective.
Sample & Assay Technologies- 4 -For Internal Use Only
Research challengeConstructing Signaling Networks
Sample & Assay Technologies- 5 -For Internal Use Only
Research challengeConstructing signaling networks
Sample & Assay Technologies- 6 -For Internal Use Only
Cell-based assays Research Challenges
■ Intra-experiment variabilityWell-to-well, plate-to-plate reproducibility
■ Inter-experiment variability
“status” of the cellswho in the lab is conducting the experiments.
■ Assay performanceSensitivity, specificity, signal to noise ratio, and simplicity
Sample & Assay Technologies- 7 -For Internal Use Only
Cignal™ Reporter AssaysFlexible, pre-optimized solution for cell based assa ys
■ Functionally validated dual-reporter formulation (L uciferase)Minimizes experimental variability, increasing biological relevance
■ Ready-to-use reporters and controls, in transfectio n ready format Enables simple, rapid analysis of the regulation of 45 signal transduction pathways
■ Genetically engineered transcriptional regulatory elements (TREs) & Destabilized , codon –optimized firefly luciferaseIncreases the signal to noise ratio, maximizes assay sensitivity and specificity
Available in both Luciferase and GFP formats
Sample & Assay Technologies- 8 -For Internal Use Only
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
Sample & Assay Technologies- 9 -For Internal Use Only
Reporter Construct GFP/firefly luciferaseTATAbox
Tandem repeats of TRE
Pathway-targeted Transcriptional Regulatory Element s (TRE), which establish the
pathway specificity of each reporter
Cignal Reporter Assays: Complete SolutionCignal™ Reporter AssaysFlexible solution: Multiple Reporter Formats
Reporter Protein is expressed when TF
binds to TRE
Sample & Assay Technologies- 10 -For Internal Use Only
Reporter Construct GFP/firefly luciferaseTATAbox
Tandem repeats of TRE
Pathway-targeted Transcriptional Regulatory Element s (TRE), which establish the
pathway specificity of each reporter
TF EGFP FL
Upstream SignalingEvents
Cignal™ Reporter AssaysFlexible solution: Multiple Reporter Formats
Sample & Assay Technologies- 11 -For Internal Use Only
Reporter Construct firefly luciferaseTATAbox
Tandem repeats of TRE
Pathway-targeted Transcriptional Regulatory Element s (TRE), which establish the
pathway specificity of each reporter
TATAboxCMVPositive Control Construct
Negative Control Construct TATAbox
Renilla LuciferaseTATAboxCMVInternal Control Construct
firefly luciferase
firefly luciferase
Cignal™ Reporter AssaysMultiple Reporter Formats – Complete dual luciferase system
Sample & Assay Technologies- 12 -For Internal Use Only
Cignal™ Reporter AssaysPlasmid - 45 Pathways
Pathway Transcription FactorAmino Acid Deprivation ATF4/ATF3/ATF2Androgen Androgen ReceptorAntioxidant Response Nrf2 & Nrf1ATF6 ATF6C/EBP C/EBP cAMP/PKA CREB Cell Cycle E2F/DP1 DNA Damage p53 EGR1 EGR1ER Stress CBF/NF-Y/YY1Estrogen ER GATA GATA Glucocorticoid GRHeat Shock Response HSFHeavy Metal Stress MTF1Hedgehog GliHNF4 HNF4Hypoxia HIF-1 Interferon Regulation IRF-1Type I Interferon STAT1/STAT2 Interferon Gamma STAT1/STAT1 KLF4 KLF4Liver X LXR
Pathway Transcription FactorMAPK/ERK Elk-1/SRF MAPK/JNK AP-1 MEF2 MEF2c-myc Myc/Max Nanog NanogNFκB NFκBNotch RBP-JkOct4 Oct4Pax6 Pax6PI3K/AKT FOXOPKC/Ca++ NFAT PPAR PPARProgesterone PRRetinoic Acid RAR Retinoid X RXRSox2 Sox2SP1 SP1STAT3 STAT3TGFβ SMAD2/3/4 VDRE Vitamin D ReceptorWnt TCF/LEFXenobiotic AhR
Sample & Assay Technologies- 13 -For Internal Use Only
Cignal™ Reporter AssaysProduct offering
Easy totransfect cell lines
Primary cells, stem cells,and difficult to transfect cell lines
EndpointAssays
EndpointAssays
Dynamic Live Cell Assays
Dynamic Live Cell Assays
Cignal Dual-Luciferase Reporter Assays
Cignal LentiLuciferase Reporters
Cignal GFP Reporter Assays
Cignal Lenti GFP Reporters
Single PathwayAssays
Single PathwayAssays
Single PathwayAssays
Single PathwayAssays
10-PathwayArrays
10-PathwayArrays
Plasmid and Lentiviral, Luciferase and GFP reporters, Single, 10-pathway, 45-pathway
Multiple options to suit your research needs:
Sample & Assay Technologies- 14 -For Internal Use Only
Cignal™ Reporter AssaysLentiviral Assays (35 Pathways)
Pathway Transcription FactorAmino Acid Deprivation ATF4/ATF3/ATF2Androgen Androgen ReceptorAntioxidant Response Nrf2 & Nrf1ATF6 ATF6C/EBP C/EBP cAMP/PKA CREB Cell Cycle E2F/DP1DNA Damage p53 EGR1 EGR1ER Stress CBF/NF-Y/YY1Estrogen ER GATA GATA Glucocorticoid GRHeat Shock Response HSFHeavy Metal Stress MTF1Hedgehog GliHNF4 HNF4Hypoxia HIF-1 Interferon Regulation IRF-1Type I Interferon STAT1/STAT2 Interferon Gamma STAT1/STAT1 KLF4 KLF4Liver X LXR
Pathway Transcription FactorMAPK/ERK Elk-1/SRF MAPK/JNK AP-1 MEF2 MEF2c-myc Myc/Max Nanog NanogNFκB NFκBNotch RBP-JkOct4 Oct4Pax6 Pax6PI3K/AKT FOXOPKC/Ca++ NFAT PPAR PPARProgesterone PRRetinoic Acid RAR Retinoid X RXRSox2 Sox2SP1 SP1STAT3 STAT3TGFβ SMAD2/3/4 VDRE Vitamin D ReceptorWnt TCF/LEFXenobiotic AhR
Sample & Assay Technologies- 15 -For Internal Use Only
Using Cignal™ Reporter AssaysWhat 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 & Assay Technologies- 16 -For Internal Use Only
Cignal™ Reporter AssaysKey advantages – Assay design- Dual Luciferase
Reporter Construct Firefly luciferaseTATAboxTRE
Renilla luciferaseTATAboxCMVNormalization Construct
Pathway-targeted transcriptionalregulatory elements (45)
Constitutive transcriptionalregulatory element (1)
The Transcriptional Regulatory Elements (TREs) esta blishes thepathway specificity of each reporter!
Sample & Assay Technologies- 17 -For Internal Use Only
Cignal™ Reporter AssaysWhy 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 Assaydesign corrects for these unwanted sources of variability.
Sample & Assay Technologies- 18 -For Internal Use Only
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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
Cignal™ Reporter AssaysDual Luciferase Assays - data
Sample & Assay Technologies- 19 -For Internal Use Only
Reporter Construct Firefly luciferaseTATAboxTRE
Renilla luciferaseTATAboxCMVNormalization Construct
Mammalian codon-optimized AND
Destabilized firefly luciferase
Cignal™ Reporter AssaysAdvantage – Genetic engineered luciferase
Sample & Assay Technologies- 20 -For Internal Use Only
Modified Luciferase:Background NFkB Reporter Signal
(no treatment post-transfection)
Modified Luciferase:NFkB Reporter Signal
Following TNF αααα Induction
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NFkB Reporter:Stable Luc
NFkB Reporter:Destabilized Luc
NegativeControl
NFkB Reporter:Stable Luc
NFkB Reporter:Destabilized Luc
Cignal™ Reporter AssaysAdvantage – Genetic engineered luciferase
High Signal to Noise RatioOffers higher sensitivity
Sample & Assay Technologies- 21 -For Internal Use Only
Reporter Construct Firefly luciferaseTATAboxTRE
Renilla luciferaseTATAboxCMVNormalization Construct
Pathway-targeted transcriptionalregulatory elements (45)
Cignal™ Reporter AssaysAdvantage – Genetic engineered TRE, optimized for eac h pathway
Sample & Assay Technologies- 22 -For Internal Use Only
TRE Maximizes Luciferase Signal
Dual-Luciferase AssaysUtilizing Cignal p53 TRE
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p53 Reporter+ DMSO
p53 Reporter+ 1µM Doxorubicin
Dual-Luciferase AssaysUtilizing Common p53 TRE
p53 Reporter+ DMSO
p53 Reporter+ 1µM Doxorubicin
125-Fold2.5-Fold
Cignal™ Reporter AssaysAdvantage – Genetic engineered TRE, optimized for eac h pathway
Sample & Assay Technologies- 23 -For Internal Use Only
Cignal™ Reporter AssaysExperimental Design
Sample & Assay Technologies- 24 -For Internal Use Only
Reporter Construct GFPTATAbox
Tandem repeats of TRE
Pathway-targeted Transcriptional Regulatory Element s (TRE), which establish the
pathway specificity of each reporter
TATAboxCMVPositive Control Construct
Negative Control Construct TATAbox GFP
GFP
Cignal™ Reporter AssaysComplete GFP system: to study interactions in indiv idual cells
Sample & Assay Technologies- 25 -For Internal Use Only
Cignal™ Reporter AssaysDNA-Vector GFP – 16 pathways
Pathway Transcription FactorAmino Acid Deprivation ATF4/ATF3/ATF2Androgen Androgen ReceptorAntioxidant Response Nrf2 & Nrf1ATF6 ATF6C/EBP C/EBPcAMP/PKA CREBCell Cycle E2F/DP1 DNA Damage p53 EGR1 EGR1ER Stress CBF/NF-Y/YY1Estrogen ER GATA GATA Glucocorticoid GRHeat Shock Response HSFHeavy Metal Stress MTF1Hedgehog GliHNF4 HNF4Hypoxia HIF-1 Interferon Regulation IRF-1Type I Interferon STAT1/STAT2Interferon Gamma STAT1/STAT1 KLF4 KLF4Liver X LXR
Pathway Transcription FactorMAPK/ERK Elk-1/SRF MAPK/JNK AP-1MEF2 MEF2c-myc Myc/Max Nanog NanogNFκB NFκBNotch RBP-JkOct4 Oct4Pax6 Pax6PI3K/AKT FOXOPKC/Ca++ NFAT PPAR PPARProgesterone PRRetinoic Acid RAR Retinoid X RXRSox2 Sox2SP1 SP1STAT3 STAT3TGFβ SMAD2/3/4 VDRE Vitamin D ReceptorWnt TCF/LEFXenobiotic AhR
Sample & Assay Technologies- 26 -For Internal Use Only
Cignal™ Reporter Assays Lenti- GFP Reporter – 6 pathways
Pathway Transcription FactorAmino Acid Deprivation ATF4/ATF3/ATF2Androgen Androgen ReceptorAntioxidant Response Nrf2 & Nrf1ATF6 ATF6C/EBP C/EBP cAMP/PKA CREB Cell Cycle E2F/DP1 DNA Damage p53 EGR1 EGR1ER Stress CBF/NF-Y/YY1Estrogen ER GATA GATA Glucocorticoid GRHeat Shock Response HSFHeavy Metal Stress MTF1Hedgehog GliHNF4 HNF4Hypoxia HIF-1 Interferon Regulation IRF-1Type I Interferon STAT1/STAT2 Interferon Gamma STAT1/STAT1 KLF4 KLF4Liver X LXR
Pathway Transcription FactorMAPK/ERK Elk-1/SRF MAPK/JNK AP-1 MEF2 MEF2c-myc Myc/Max Nanog NanogNFκB NFκBNotch RBP-JkOct4 Oct4Pax6 Pax6PI3K/AKT FOXOPKC/Ca++ NFAT PPAR PPARProgesterone PRRetinoic Acid RAR Retinoid X RXRSox2 Sox2SP1 SP1STAT3 STAT3TGFβ SMAD2/3/4 VDRE Vitamin D ReceptorWnt TCF/LEFXenobiotic AhR
Sample & Assay Technologies- 27 -For Internal Use Only
Treatment of Cignal SRE (GFP) transfectants with 10% serum and 10 ng/ml PMAactivates the MAPK/ERK signaling pathway.
FluorometryFluorescent Microscopy
Cignal™ Reporter AssaysGFP system application: Small molecule studies
Sample & Assay Technologies- 28 -For Internal Use Only
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
Sample & Assay Technologies- 29 -For Internal Use Only
Functional GenomicsWhat’s the phenotype of my gene?Does my gene of interest regulate signaling pathway “X”?
Functional ProteomicsWhat’s the phenotype of my protein/peptide?Does my protein, peptide, or growth factor of interest regulate signaling pathway “X”?
Drug ScreeningWhat is the mechanism of action of my small molecule drug candidates?Do my drug candidates of interest regulate signaling pathway “X”?
Cignal™ Reporter AssaysApplication in multiple research areas
Sample & Assay Technologies- 30 -For Internal Use Only
Cignal™ Reporter AssaysApplication – RNA Interference
Sample & Assay Technologies- 31 -For Internal Use Only
Functional GenomicsWhat’s the phenotype of my gene?Does my gene of interest regulate signaling pathway “X”?
Functional ProteomicsWhat’s the phenotype of my protein/peptide? Does my protein, peptide, or growth factor of interest regulate signaling pathway “X”?
Drug ScreeningWhat is the mechanism of action of my small molecule drug candidates? Do my drug candidates of interest regulate signaling pathway “X”?
Cignal™ Reporter AssaysApplication in multiple research areas
Sample & Assay Technologies- 32 -For Internal Use Only
Cignal™ Reporter AssaysApplication – Functional Proteomics
Sample & Assay Technologies- 33 -For Internal Use Only
Functional GenomicsWhat’s the phenotype of my gene?Does my gene of interest regulate signaling pathway “X”?
Functional ProteomicsWhat’s the phenotype of my protein/peptide? Does my protein, peptide, or growth factor of interest regulate signaling pathway “X”?
Drug ScreeningWhat is the mechanism of action of my small molecule drug candidates? Do my drug candidates of interest regulate signaling pathway “X”?
Cignal™ Reporter AssaysApplication in multiple research areas
Sample & Assay Technologies- 34 -For Internal Use Only
Cignal™ Reporter AssaysApplication – Small Molecule Studies
Sample & Assay Technologies- 35 -For Internal Use Only
SituationI have a biological molecule that knocks down p53 expression
Question1. 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 cancer-relevant signaling pathways?
Cignal reporter assaysApplication – Study pathway interactionResearch challengePathway Interaction Study
Sample & Assay Technologies- 36 -For Internal Use Only
Wnt Signaling NFkB Signaling
E2F Signaling
MAPK/ERK Signaling
Myc Signaling
MAPK/JNK Signaling
TGFβ Signaling
Notch Signaling
?
?
?
?
?
?
?
?
?Hypoxia Signaling
p53 Signaling
Research challengePathway interactions
Sample & Assay Technologies- 37 -For Internal Use Only
Approach:Carry out a p53 RNA interference experiment with multiple cancer-relevant Cignal Reporter Assays
Cignal™ Reporter Assays Application – study pathway interaction
Sample & Assay Technologies- 38 -For Internal Use Only
Cignal Finder arraysCancer array design
Pathway
Cell CycleDNA DamageHypoxiaMAPK/ERKMAPK/JNKc-MycNFkBNotchTGFβWnt
Transcription Factor
E2F/DP1p53HIFElk-1/SRFAP1Myc/MaxNFkBRBP-JkSMAD2/SMAD3/SMAD4TCF/LEF
Sample & Assay Technologies- 39 -For Internal Use Only
Cignal Finder arrayHow does it work?
Sample & Assay Technologies- 40 -For Internal Use Only
Cignal Finder cancer array p53 RNAi study design
1 2 3 4 5 6 7 8 11
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
10. HIF Cignal Reporter
11. Cignal Negative Control
12. Cignal Positive Control
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p53 siRNA
Neg. control siRNA
Sample & Assay Technologies- 41 -For Internal Use Only
Impact of p53 siRNA Treatment
Cignal Finder cancer array p53 RNAi study design - results
Sample & Assay Technologies- 42 -For Internal Use Only
MAPK/ERK Signaling
Notch Signaling
Hypoxia Signaling
p53 Signaling
Cignal Finder cancer array p53 RNAi study design – next steps
Sample & Assay Technologies- 43 -For Internal Use Only
Cignal™ Reporter Assays SystemSummary
■ 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 ass ays
■ Dual Modalities■ Dual-luciferase quantitative endpoint assays■ GFP dynamic live cell assays with single cell resolution
Sample & Assay Technologies
New research tool for 2013
http://www.sabiosciences.com/sciglobe.php
Sample & Assay Technologies- 45 -For Internal Use Only
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: [email protected]
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
Thank you!Krishnan [email protected]