Cignal pathway reporter assays provide a rapid, sensitive, and quantitative assessment of signal transduction pathway activation by measuring the activities of downstream transcription factors. Each Cignal pathway reporter assay kit is a dual-luciferase assay and includes a pathway-focused transcription factor reporter, a non-inducible negative control, as well as positive control luciferase and positive control GFP. These assays are powerful and useful tools in functional genomics and drug discovery for assessing the biological impact of siRNAs, proteins, and small molecule compounds.

Product Listing and Performance Data

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Why Cignal Reporter Assays?

• Reproducibility
  Functionally validated dual reporter formulation minimizes variability, increasing the biological relevance of each experiment.
• Versatility and Sensitivity
  Proprietary TRE structure allows monitoring both up- and down-regulations. Use of destabilized luciferase increases the signal to noise ratio, maximizing assay sensitivity.
• Convenience
  Transfection-ready easy-to-use assay kits, including positive and negative controls, coupled with transient reporter system, enable rapid analysis of signal transduction pathway regulation.

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 results are calculated for each transfectant. The impact of the experimental treatments is determined by comparing the normalized luciferase activities of the reporter assay transfectants to the identically treated negative control transfectants, across the complete treatment regimen. 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.

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 NF-κB 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.