cignal webina
TRANSCRIPT
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Cignal™ Reporter Assays
Simple and Robust Signal Transduction Assays
Brian McNally, Ph.D. Associate Product Manager
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Topics to be Covered
• Who is SABiosciences?
• 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)
• How YOU Can Use Cignal Reporter Assays• Performance and applications data.
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Who IS SABiosciences?
• Located in Frederick, Maryland, USA (near to NIH Bethesda campus)
• Founded 10 years ago by scientists working at the NIH• Core Values: Innovation, Focus, Systematic Design• Goal: Transform cutting-edge biological information and
technologies into easy to use, accessible, and innovative systems biology research tools.
• Products: Gene expression analysis, protein function analysis and cell based assays
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Systems Biology @ SABiosciences
DNA
RNA
Protein
SequencingSNP DetectionChIPMethylation
ChIP-qPCR AssaysMethyl Profiler
Discovery Screening
Confirmation
Gene Expression Service CoreRT2 PCR ArraysRT2 miRNA Arrays and AssaysRT2 qPCR Assays
DetectionKnockdownReporter SystemPhenotypes
ELISArray KitssiRNA Arrays / shRNA PlasmidsCignal Reporter SystemCASE Kits
Genomics &Regulation
Gene Expression
Gene FunctionProtein Function
Technologies SABiosciences ProductsResearch
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Topics to be Covered• Who is SABiosciences?
• 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)
• How YOU Can Use Cignal Reporter Assays• Performance and applications data.
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Cignal Applications
• Monitoring signaling pathway activationIs my pathway of interest regulated by biological molecule “X”?
• Studying the biological response of a drug candidate or chemical compound Does my compound of interest regulate pathway “X”?
• Phenotypic analysis of gene knockdown or gene over-expressionDoes my gene of interest regulate pathway “X”?
• Mode of action of protein, peptide and ligandsDoes my protein/peptide/ligand of interest regulate pathway “X”?
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Cell-based Assay Research Challenges
• Variability within experimentsWell-to-well and plate-to-plate reproducibility are notoriously challenging with cell-based assays.
• Variability between experimentsReliable results often depend upon the “status” of the cells, and who in the lab is conducting the experiments.
• Assay PerformanceSensitivity, specificity, signal to noise ratio, and simplicity are of utmost importance.
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What Cignal Reporter Assays Provide
• Functionally validated dual-reporter formulation Minimizes variability, increasing the biological relevance of each experiment
• Ready-to-use reporters and controls, coupled with a transient reporter systemEnables simple and rapid analysis of the regulation of 29 signal transduction pathways
• Combination of custom-engineered transcriptional regulatory elements (TREs) and destabilized, codon- optimized firefly luciferaseIncreases the signal to noise ratio and maximizes assay sensitivity and specificity
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Topics to be Covered
• Who is SABiosciences?• 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)
• How YOU Can Use Cignal Reporter Assays• Performance and applications data.
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Cignal Pathway Reporter System Products
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-LuciferaseReporter Assays
Cignal LentiLuciferase Reporters
Cignal GFP Reporter Assays
Cignal LentiGFP Reporters
Single PathwayAssays
Single PathwayAssays
Single PathwayAssays
Single PathwayAssays
10-PathwayArrays
10-PathwayArrays
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Cignal Reporter AssaysPathway Transcription FactorAmino Acid Deprivation ATF4/ATF3/ATF2Androgen Androgen ReceptorAntioxidant Response Nrf2 & Nrf1ATF6 ATF6C/EBP C/EBPcAMP/PKA CREB Cell Cycle E2F/DP1 DNA Damage p53 EGR1 EGR1ER Stress CBF/NF-Y/YY1Estrogen ER GATA GATAGlucocorticoid GRHeat Shock Response HSFHeavy Metal Stress MTF1Hedgehog GLIHypoxia HIF-1 Type I Interferon STAT1/STAT2 Interferon Gamma STAT1/STAT1
Pathway Transcription FactorMAPK/ERK Elk-1/SRF MAPK/JNK AP-1 MEF2 MEF2c-myc Myc/Max NFκB NFκBNotch RBP-JkOct4 Oct4PI3K/AKT FOXOPKC/Ca++ NFAT PPAR PPARRetinoic Acid RAR SP1 SP1STAT3 STAT3TGFβ
SMAD2/3/4 VDRE Vitamin D ReceptorWnt TCF/LEF
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REPORTER DESIGN: Dual-Luciferase Assays
Reporter Construct Firefly luciferaseTATAboxTRE
Renilla luciferaseTATAboxCMVNormalization Construct
Pathway-targeted transcriptionalregulatory elements (35)
Constitutive transcriptionalregulatory element (1)
The Transcriptional Regulatory Elements (TREs) are what establish the
pathway specificity of each reporter!
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Using Cignal Reporters: What You Need
• Cignal Reporter Assay
• Reliable transfection reagent and protocol (SureFECT)
• Test Biological• siRNA (SureSilencing siRNA Arrays)• shRNA (SureSilencing shRNA)• expression vectors• protein/peptide• small molecule
• Detection Instrument and Luciferase Substrates
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REPORTER DESIGN: Dual-Luciferase Assays
Reporter Construct Firefly luciferaseTATAboxTRE
Renilla luciferaseTATAboxCMVNormalization Construct
Pathway-targeted transcriptionalregulatory elements (35)
Constitutive transcriptionalregulatory element (1)
The Transcriptional Regulatory Elements (TREs) are what establish the
pathway specificity of each reporter!
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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 Assaydesign corrects for these unwanted sources of variability.
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tsDual Luciferase Format Minimizes Variability
Dual-Luciferase Assays:NFkB Reporter Assay
CV=0.09
CV=0.07
Single-Luciferase Assays:NFkB Reporter Assay
NFkB Reporter+ NFkB siRNA
CV=0.69
CV=0.70
NFkB Reporter+ Neg. control siRNA
NFkB Reporter+ NFkB siRNA
NFkB Reporter+ Neg. control siRNA
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REPORTER DESIGN: Dual-Luciferase Assays
Reporter Construct Firefly luciferaseTATAboxTRE
Renilla luciferaseTATAboxCMVNormalization Construct
Mammalian codon-optimized AND
Destabilized firefly luciferase
The Transcriptional Regulatory Elements (TREs) are what establish the
pathway specificity of each reporter!
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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
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WHY?
NegativeControl
NFkB Reporter:Stable Luc
NFkB Reporter:Destabilized Luc
NegativeControl
NFkB Reporter:Stable Luc
NFkB Reporter:Destabilized Luc
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REPORTER DESIGN: Dual-Luciferase Assays
Reporter Construct Firefly luciferaseTATAboxTRE
Renilla luciferaseTATAboxCMVNormalization Construct
Pathway-targeted transcriptionalregulatory elements (35)
The Transcriptional Regulatory Elements (TREs) are what establish the
pathway specificity of each reporter!
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Cignal TRE Maximize 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
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Cignal Reporter Assays: How It Works
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Cignal Reporter Assays (GFP)Pathway Transcription FactorAmino Acid Deprivation ATF4/ATF3/ATF2Androgen Androgen ReceptorAntioxidant Response Nrf2 & Nrf1ATF6 ATF6C/EBP C/EBPcAMP/PKA CREB Cell Cycle E2F/DP1 DNA Damage p53 EGR1 EGR1ER Stress CBF/NF-Y/YY1Estrogen ER GATA GATAGlucocorticoid GRHeat Shock Response HSFHeavy Metal Stress MTF1Hedgehog GLIHypoxia HIF-1 Type I Interferon STAT1/STAT2 Interferon Gamma STAT1/STAT1
Pathway Transcription FactorMAPK/ERK Elk-1/SRF MAPK/JNK AP-1MEF2 MEF2c-myc Myc/MaxNFκB NFκBNotch RBP-JkOct4 Oct4PI3K/AKT FOXOPKC/Ca++ NFAT PPAR PPARRetinoic Acid RAR SP1 SP1STAT3 STAT3TGFβ
SMAD2/3/4VDRE Vitamin D ReceptorWnt TCF/LEF
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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)
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Cignal Finder Arrays: Cancer Array Design
Pathway
Cell CycleDNA DamageHypoxiaMAPK/ERKMAPK/JNKc-MycNFkBNotchTGFβWnt
Transcription Factor
E2F/DP1p53HIFElk-1/SRFAP1Myc/MaxNFkBRBP-JkSMAD2/SMAD3/SMAD4TCF/LEF
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How Does a Cignal Finder Array Work?
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Topics to be Covered
• Who is SABiosciences?• 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)
• How YOU Can Use Cignal Reporter Assays• Performance and applications data
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Cignal Applications
• 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”?
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Cignal Application: RNA Interference
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Cignal Reporter:
siRNA:
NC NC
NC siRNA
p53 p53
NC siRNAp53 siRNA p53 siRNA
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Cignal Applications
• 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”?
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Cignal Application: Functional Proteomics
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Cignal Reporter:
Protein:
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NC NC SMAD SMAD SMAD
None None25 ng/µlhuTGFβ1
2.5 ng/mlhuTGFβ1
25 ng/mlhuTGFβ1
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Cignal Applications
• 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”?
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Cignal Application: Small Molecule Studies
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Cignal Reporter: CRE CRE CRE CRE
Small Molecule: None 0.1µM Forskolin
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200
250
CRE
1µM Forskolin
5µM Forskolin
10µM Forskolin
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Cignal Application(GFP): Small Molecule Studies
Treatment of Cignal SRE (GFP) transfectants with 10% serum and 10 ng/ml PMAactivates the MAPK/ERK signaling pathway.
Fluorometry Fluorescent Microscopy
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Application Study
Question:What is impact of p53 gene expression knock down on the p53 signaling pathway AND other cancer-relevantsignaling pathways?
Approach:Carry out a p53 RNA interference experiment with multiple cancer-relevant Cignal Reporter Assays.
p53 Pathway Interaction Study
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Post-Genomics Research Challenge
Signal TransductionCascadeA B C D
(~40 proteins)
p53 Signaling
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Cignal Reporters: Constructing Signaling Networks
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Post-Genomics Research Challenge: Pathway Interactions?
Wnt Signaling NFkB Signaling
E2F Signaling
MAPK/ERK Signaling
Myc Signaling
MAPK/JNK Signaling
TGFβ Signaling
Notch Signaling
?
?
?
?
?
?
?
?
?Hypoxia Signaling
p53 Signaling
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Cignal Finder Cancer Array : p53 RNAi Study
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
129 10
p53 siRNA
Neg. control siRNA
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Results: Cancer Array Application Study
00.5
11.5
22.5
33.5
44.5
TCF/L
EFRBP-Jk
p53
SMADpR
B-E2F
NFkB Myc HIF
SRE
AP1
Cignal Pathway Reporter
Fold
Cha
nge
in P
athw
ayA
ctiv
atio
n
Impact of p53 siRNA Treatment
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Post-Genomics Research Challenge: Pathway Interactions?
MAPK/ERK Signaling
Notch Signaling
Hypoxia Signaling
p53 Signaling
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Topics to be Covered
• Who is SABiosciences?• 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)
• How YOU Can Use Cignal Reporter Assays• Performance and applications data
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Summary: Cignal Reporter Assay System• Breadth of Pathways and Research Applications
•35 Pathway-Focused Reporter Assays•4 Application-Specific Cignal Finder 10-Pathway Arrays
• PerformanceExceptional Sensitivity, Specificity, Signal-to-Noise Ratio, and Reproducibility
• ConvenienceReady-to-use, validated, preformatted transient assays
• Dual Modalities•Dual-luciferase quantitative endpoint assays•GFP dynamic live cell assays with single cell resolution
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Cignal Reporter Assays
Questions?Contact Technical Support 9 AM – 6 PM Eastern M – F
Telephone: 888-503-3187; Email: [email protected]
Simple and Robust Signal Transduction Assays