The Cignal Reporter Assay System provide a rapide, sensitive and quantitative assessment of signal transduction pathway activation by measuring the activities of downstream transcription factors, using either dual-luciferase or green fluorescent protein (GFP) reporter systems.
 

Single Cignal dual-luciferase or GFP Reporter Assays

• The dual-luciferase format is an endpoint assay providing unsurpassed sensitivity, specificity, and signal-to-noise ratios. This format is available for all 18 Cignal Reporter Assays. Each assay includes a pathway-focused transcription factor reporter, a non-inducible negative control, as well as luciferase and GFP positive controls.

• The GFP format is an outstanding method for monitoring live cell pathway regulation, with single cell resolution. This format is currently available for six of the Cignal Reporter Assays. It includes the pathway-focused transcription factor reporter, and the necessary negative and positive controls.

How it works

The Cignal Reporter Assays include pre-formulated, transfection-ready pathway reporter, negative control, and positive control. The inducible pathway reporter and non-inducible negative control are transfected and subjected to experimental treatments, in parallel.

Dual-luciferase: The Cignal Reporter Assays (luc) include pre-formulated, transfection-ready reporter, negative control, and positive control. The transcription factor reporter and negative control are transfected and subjected to experimental treatments, in parallel. Dual-luciferase results are calculated for each transfectant. The change in the activity of each signaling pathway is determined by comparing the normalized luciferase activities of the reporter in treated versus untreated transfectants. The identically treated negative control transfectants serve as a specificity control. The positive control serves as a control for transfection efficiency, by monitoring GFP expression, as well as a positive control for both the firefly and Renilla luciferase assays.

 

GFP: The Cignal Reporter Assays (GFP) include pre-formulated, transfection-ready reporter, negative control, and positive control. The transcription factor reporter and negative control are transfected and subjected to experimental treatments, in parallel. GFP expression is quantitated using a flow cytometer, fluorescent microscope, or fluorometer. The change in the activity of each signaling pathway is determined by comparing the GFP activities in treated versus untreated transfectants. The identically treated negative control transfectants serve as a specificity control. The positive control serves as a control for transfection efficiency, by monitoring GFP expression from the constitutively expressing CMV-GFP reporter.




 

Dual-Luciferase Applications Data

• Functional Genomics: Assessing RNA Interference Phenotypes

Cignal p53 Reporter Assays showed that p53 siRNA treatment abolished p53 transcription activity
HCT 116 cells were transfected with p53 reporter, negative control and positive control along with p53 siRNA or negative control siRNA. Dual Luciferase assays were performed, and promoter activity values are expressed as arbitrary units using a Renilla reporter for internal normalization. Experiments were done in triplicates, and the standard deviation is indicated.




 

• Functional Genomics: Assessing Overexpression Phenotypes

Cignal RBP-Jk Reporter showed up-regulation of Notch signaling activity after over expression of activated Notch1
293 H cells were transfected with RBP-Jk reporter, negative control and positive control. After 24 hours of transfection, cells were infected with 100 MOI of recombinant adenoviruses expressing activated Notch1 (Ad-NICD) or 100 MOI of recombinant adenovirus expressing GFP (Ad-GFP) for another 18 hours. Dual Luciferase assays were performed, and promoter activity values are expressed as arbitrary units using a Renilla reporter for internal normalization. Experiments were done in triplicates, and the standard deviation is indicated.



 

 

• Functional Proteomics: Analyze the Effects of Recombinant Protein or Peptide Treatments

Cignal NFκB reporter showed that Human Tumor Necrosis Factor Alpha (TNFα) activated NFκB signaling activity in a dose-dependent manner
293 H cells were transfected with NFκB reporter, negative control and positive control (for transfection protocol refer our user manual). After 24 hours of transfection, cells were treated with different doses of hTNFα for another 24 hours. Dual Luciferase assays were performed, and promoter activity values are expressed as arbitrary units using a Renilla reporter for internal normalization. Experiments were done in triplicates, and the standard deviation is indicated.










 

• Drug Discovery: Screen and Validate Small Molecule Drug Candidates

Cignal RARE reporter assay reported elevated retinoic acid receptor pathway activity after the treatment of trans-retinoic acid (ATRA)
CHO-K1 cells were transfected with RARE reporter, negative control and positive control (for transfection protocol refer to our user manual). After 16 hours of transfection, medium was changed to assay medium (Opti-MEM + 1% charcoal stripped FBS + 0.1mM NEAA + 1mM Sodium pyruvate + 100 μg/ml penicillin + 100 µg/ml streptomycin). After 24 hours of transfection the cells were treated with 1µM all trans-rectinoic acid (ATRA) for 6 hours. Dual Luciferase assay was performed, and promoter activity values are expressed as arbitrary units using a Renilla reporter for internal normalization. Experiments were done in triplicates, and the standard deviation is indicated.