Cignal™ Reporter Assays

Cignal Reporter Assays provide a rapid, sensitive, and quantitative assessment of signal transduction pathway activation by measuring the activities of downstream transcription factors. Two reporter systems are available.

• The dual-luciferase format is an endpoint assay providing unsurpassed sensitivity, specificity, and signal-to-noise ratios. This format is available for all 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.

These assays are powerful tools in functional genomics and drug discovery for assessing the biological impact of siRNAs, proteins, and small molecule compounds.

Product Listing and Performance Data

Click here to view the Transcriptional Response Element (TRE) sequences for each reporter assay.

Why Cignal Reporter Assays?

• DUAL-LUCIFERASE FORMAT:
  Exceptional sensitivity, reproducibility, specificity, and signal to noise ratio
  
• GFP FORMAT:
  Single cell resolution; Real-time live cell quantification
  
• CONVENIENCE:
  Transfection-ready constructs, 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: 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.

GFP Application Data

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

Fluorescence Microscopy: Cignal NFκB GFP reporter showed that Human Tumor Necrosis Factor Alpha (hTNFα) activated the NFκB signaling pathway

293-H cells were transfected with the Cignal NFκB-GFP reporter or negative control (for transfection protocol refer our user manual). After 16 hours of transfection, medium was changed to assay medium (Opti-MEM + 0.5% FBS + 0.1mM NEAA + 1mM Sodium pyruvate + 100 U/ml penicillin + 100 µg/ml streptomycin). After 24 hours of transfection, cells were treated with 10 ng/ml hTNF . After 18 hours of treatment, bright field and fluorescent images were taken of the cultures transfected with the negative control reporter (A and B, respectively) and the Cignal NFκB-GFP reporter (C and D, respectively).

Quantitative Fluorometry: Cignal NFκB GFP reporter showed that Human Tumor Necrosis Factor Alpha (hTNFα) activated the NFκB signaling pathway

293-H cells were transfected with the Cignal NFκB-GFP reporter or negative control (for transfection protocol refer our user manual). After 16 hours of transfection, medium was changed to assay medium (Opti-MEM + 0.5% FBS + 0.1mM NEAA + 1mM Sodium pyruvate + 100 U/ml penicillin + 100 µg/ml streptomycin). After 24 hours of transfection, cells were treated with 10 ng/ml TNFα. After 18 hours of treatment, medium was removed from the wells and fluorescence activity was measured using a fluorometer. The fluorescent activity present in treated and non-treated negative control wells was subtracted from the fluorescent activity in treated and non-treated Cignal NFκB-GFP reporter wells and relative fluorescence activities are expressed as arbitrary units. Experiments were done in triplicates, and the standard deviations are indicated.

• Drug Discovery: Screen and Validate Small Molecule Drug Candidates

Fluorescence Microscopy: Cignal SRE-GFP reporter measures activation of serum response factor (SRF) transcription activity

293-H cells were transfected with the Cignal SRE-GFP reporter or negative control (for transfection protocol refer our user manual). After 16 hours of transfection, medium was changed to assay medium (Opti-MEM + 0.5% FBS + 0.1mM NEAA + 1mM Sodium pyruvate + 100 U/ml penicillin + 100 µg/ml streptomycin). After 24 hours of transfection, cells were treated with 10 ng/ml PMA and 10% serum. After 18 hours of treatment, bright field and fluorescent images were taken of the cultures transfected with the negative control reporter (A and B, respectively) and the Cignal SRE-GFP reporter (C and D, respectively).

Quantitative Fluorometry: Cignal SRE-GFP reporter measures activation of serum response factor (SRF) transcription activity

293-H cells were transfected with the Cignal SRE-GFP reporter or negative control (for transfection protocol refer our user manual). After 16 hours of transfection, medium was changed to assay medium (Opti-MEM + 0.5% FBS + 0.1mM NEAA + 1mM Sodium pyruvate + 100 U/ml penicillin + 100 µg/ml streptomycin). After 24 hours of transfection, cells were treated with 10 ng/ml PMA and 10% serum. After 18 hours of treatment, medium was removed from the wells and fluorescence activity was measured using a fluorometer. The fluorescent activity present in treated and non-treated negative control wells was subtracted from the fluorescent activity in treated and non-treated Cignal SRE-GFP reporter wells and relative fluorescence activities are expressed as arbitrary units. Experiments were done in triplicates, and the standard deviations are indicated.