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Cignal Reporter Assay Kits

For rapid, sensitive, and quantitative assessment of signal transduction pathway activation

  • Rapid analysis of signal transduction pathway regulation
  • Exceptional sensitivity and specificity
  • Transfection-ready constructs
  • Includes positive and negative controls
  • Real-time live cell quantification (GFP format)

Cignal Reporter Assays provided in the Cignal Reporter Assay Kit enable rapid, sensitive, and quantitative assessment of signal transduction pathway activation by measuring the activities of downstream transcription factors. Two reporter systems are available: dual-luciferase format and GFP format.

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Cignal Reporter Assay Kits are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.


0
TNFα activates NFkB signaling activity in a dose-dependent manner.
293 H cells were transfected with Cignal NFkB Reporter Assay, negative or positive control. After 24 hours, cells were treated with different doses of hTNFα for a further 24 hours. Dual-luciferase assays were performed, and reporter activity values are expressed as arbitrary units using a Renilla reporter for internal normalization. Experiments were performed in triplicate and standard deviation is shown.
1
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, together with p53 siRNA or negative control siRNA. Dual-luciferase assays were performed. Reporter activity values are expressed as arbitrary units using a Renilla reporter for internal normalization. Experiments were performed in triplicate and the standard deviation is shown.

2
Assessing overexpression phenotypes.

Cignal RBP-Jκ Reporter Assay showed upregulation of Notch signaling activity after overexpression of activated Notch1. 293 H cells were transfected with RBP-Jκ reporter, negative control, and positive control. After 24 hours, 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. Reporter activity values are expressed as arbitrary units using a Renilla reporter for internal normalization. Experiments were performed in triplicate and the standard deviation is shown.

3
Verification of small molecule drug candidate.

Cignal Retinoic Acid Response Element (RARE) Reporter Assay reported elevated retinoic acid receptor pathway activity after the treatment of all trans-retinoic acid (ATRA). CHO-K1 cells were transfected with RARE reporter, negative control, and positive control. After 16 hours, medium was changed to assay medium. After 24 hours, cells were treated with 1 µM 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 performed in triplicate and the standard deviation is shown.

4
Cignal Reporter Assay shows that hTNFa activated NFkB signaling pathway.
293 H cells were transfected with the Cignal NFkB-GFP Reporter Assay or negative control. After 16 hours, medium was changed to assay medium. After 24 hours, cells were treated with 10 ng/ml TNFα. After 18 hours, medium was removed from the wells and fluorescence activity was measured using a fluorometer. The fluorescence activity present in treated and nontreated negative control wells was subtracted from the fluorescence activity in treated and nontreated Cignal NFkB-GFP Reporter Assay wells and relative fluorescence activities expressed as arbitrary units. Experiments were performed in triplicate and the standard deviations are shown.
5
Cignal Reporter Assay measures activation of serum response factor (SRF) transcription activity.

293 H cells were transfected with the Cignal SRF-GFP Reporter Assay or negative control. After 16 hours, medium was changed to assay medium. After 24 hours, cells were treated with 10 ng/ml PMA and 10% serum. After 18 hours, medium was removed from the wells and fluorescence activity was measured using a fluorometer. The fluorescence activity present in treated and nontreated negative control wells was subtracted from the fluorescence activity in treated and nontreated Cignal SRF-GFP Reporter Assay wells and relative fluorescence activities expressed as arbitrary units. Experiments were performed in triplicate and the standard deviations are shown.

6
Cignal Reporter Assay procedure.
7
Cignal Reporter Assay principle.
Performance
Principle

Cignal Reporter Assays consist of multiple repeats of a specific transcription factor’s binding site and basic promoter elements to drive the expression of a reporter gene (firefly luciferase or GFP). These reporters are powerful tools in functional genomics and drug discovery for assessing pathway activity. When the pathway is activated or inhibited by a drug candidate, gene knockdown, overexpression, or peptide, luciferase or GFP reporter activity is modulated and can be measured quantitatively and rapidly (see figure "Cignal Reporter Assay principle"). The Cignal Reporter Assays are available as transfection-ready constructs utilizing two reporter systems: the dual-luciferase reporter or the GFP reporter (see flowchart "Cignal Reporter Assay procedure").

The dual-luciferase format is an end-point 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 and a noninducible 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. It includes a pathway-focused transcription factor reporter and positive and negative controls.

Procedure

Cignal Reporter Assays include a preformulated, transfection-ready pathway reporter (dual-luciferase or GFP), plus a positive and negative control. The inducible pathway reporter and noninducible negative control are transfected and subjected to experimental treatments in parallel.

Dual-luciferase

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 firefly luciferase and Renilla expression.

GFP

GFP expression is quantified using a flow cytometer, fluorescence 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.

Applications

Cignal Reporter Assays are powerful tools in functional genomics, proteomics, and drug discovery for assessing the biological impact of pathway inhibition or activation with siRNAs, proteins, and small molecule compounds.

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Brochures & Guides (1)
For cell-based analysis of pathway signaling activity
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Kit Handbooks (1)
Transfection Protocols (2)
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MTF1 Reporter Assay Kit Cignal Reporter Assay Kits
Introduction
The Cignal MTF1 Reporter Assay Kit is designed to measure signaling induced by heavy metal stress via the transcriptional activity of MTF1. The zinc finger transcription factor MTF-1 plays a central role in the metal-inducible transcriptional activation of metallothionein and other genes involved in metal homeostasis and cellular stress response. The MTF1 reporter is a mixture of a MTF1-responsive luciferase construct and a constitutively expressing Renilla luciferase construct (40:1). The GLI-responsive luciferase construct encodes the firefly luciferase reporter gene under the control of a minimal (m)CMV promoter and tandem repeats of the MTF1 transcriptional response element. The number of response elements and the intervening sequence between these response elements has been experimentally optimized to maximize the signal to noise ratio. The MTF1 reporter construct monitors both increases and decreases in the transcriptional activity of MTF1, and hence the activity of the heavy metal stress signaling pathway. The constitutively expressing Renilla luciferase construct encodes the Renilla luciferase reporter gene under the control of a CMV immediately early enhancer/promoter and acts as an internal control for normalizing transfection efficiencies and monitoring cell viability. Using a simple dual-luciferase assay, you can easily monitor the activity of MTF1 and determine the effect of various treatments, such as gene knockdown, over-expression, and chemical compounds.
CCS-5033L

The Cignal MTF1 Reporter Assay shows MTF1 transcription activity. HeLa cells were transfected with MTF1 reporter, negative control, and positive control (for the transfection protocol, refer to the Cignal Reporter Assay Handbook). After 16 hours of transfection, medium was changed to assay medium (DMEM + 0.5% FBS + 0.1 mM NEAA + 1 mM sodium pyruvate). After 40 hours of transfection, cells were treated with either 100 µM ZnSO4 or 100 µM ZnCl2 for 4 hours. A dual-luciferase assay was performed 44 hours after transfection, and promoter activity values are expressed as arbitrary units using a Renilla luciferase reporter for internal normalization. Experiments were performed in triplicate, and the standard deviation is indicated.
Name Cignal MTF1 Reporter (luc) Kit (CCS-5033L)
Official symbol Mtf1 [Mouse]
Official name metal response element binding transcription factor 1
Species Mouse (Mus musculus)
Entrez Gene ID 17764
Transcript(s) for this gene NM_008636 (7631 bp), XM_622562 (2892 bp), ENSMUST00000147452, XM_006502830 (4827 bp), XM_006502831 (4551 bp), XM_006502832 (4549 bp)
Official symbol MTF1 [Human]
Official name metal-regulatory transcription factor 1
Species Human (Homo sapiens)
Entrez Gene ID 4520
Transcript(s) for this gene NM_005955 (7986 bp), ENST00000468190, ENST00000373036
Official symbol Mtf1 [Rat]
Official name metal-regulatory transcription factor 1
Species Rat (Rattus norvegicus)
Entrez Gene ID 362591
Transcript(s) for this gene NM_001108677 (5300 bp), XM_001055855 (2709 bp), XM_342910 (2781 bp), XM_006238883 (7674 bp), XM_006238884 (7098 bp)