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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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RXR Reporter Assay Kit Cignal Reporter Assay Kits
Introduction
The Cignal RXR Reporter Assay Kit measures the activity of Retinoid X Receptor (RXR)-mediated signal transduction pathway. RXR is a member of the nuclear hormone receptor family. RXR plays an important role in many fundamental biological processes such as reproduction, cellular differentiation, bone development, hematopoesis and pattern formation during embryogenesis. RXR is also implicated in some pathological conditions as neoplastic formation and is a potential target for cancer therapy. Besides acting as a homodimer, RXR plays a central role in regulating the activity of other nuclear hormone receptors by acting as a heterodimeric partner. RXR forms functional heterodimers with retinoic acid receptor (RAR), thyroid hormone receptor, vitamin D receptor, NGFI-B and many other nuclear receptors. The RXR reporter is a mixture of an RXR-responsive luciferase construct and a constitutively expressing Renilla element (40:1). The RXR-responsive luciferase construct encodes the firefly luciferase reporter gene under the control of a minimal (m)CMV promoter and tandem repeats of the RXR transcriptional response element. The number of response elements as well as the intervening sequence between response elements have been experimentally optimized to maximize the signal to noise ratio. This reporter monitors both increases and decreases in the transcriptional activity of RXR. The constitutively expressing Renilla element 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, scientists easily monitor the activity of RXR and determine the effect of various treatments, such as gene knockdown, over-expression, and chemical compounds on RXR.
CCS-9044L

The Cignal RXR Reporter Assay shows RXR transcription activity. CHO-K1 cells were reverse transfected with the Cignal RXR Reporter, as well as with positive and negative control reporters. After 16 hours of transfection, medium was changed to assay medium (DMEM + 1% charcoal-stripped FBS + 0.1 mM NEAA). After 24 hours of transfection, cells were treated with 1 µM or 5 µM all-trans-retinoic acid (ATRA) for 18 hours. A dual-luciferase assay was performed after 42 hours of transfection, 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 indicated.
Related Biologies
Drug Metabolism
Name Cignal RXR Reporter (luc) Kit (CCS-9044L)
Official symbol RXRA [Human]
Official name retinoid X receptor, alpha
Species Human (Homo sapiens)
Entrez Gene ID 6256
Transcript(s) for this gene NM_002957 (5530 bp), ENST00000484822, NM_001291920 (6202 bp), NM_001291921 (5993 bp), XM_005263409 (2397 bp), ENST00000481739
Official symbol Rxra [Mouse]
Official name retinoid X receptor alpha
Species Mouse (Mus musculus)
Entrez Gene ID 20181
Transcript(s) for this gene NM_011305 (5267 bp), XM_976705 (1727 bp), XM_990117 (5042 bp), ENSMUST00000148756, NM_001290481 (5088 bp), NM_001290482 (5125 bp), XM_006497801 (4671 bp), XM_006497802 (4672 bp), XM_006497803 (4672 bp), XM_006497805 (4718 bp), XM_011239037 (4749 bp), XM_011239038 (4671 bp), XM_011239039 (4671 bp), XM_011239040 (4671 bp), XM_011239041 (4671 bp), XM_011239042 (4671 bp)
Official symbol Rxra [Rat]
Official name retinoid X receptor alpha
Species Rat (Rattus norvegicus)
Entrez Gene ID 25271
Transcript(s) for this gene NM_012805 (1404 bp)
Official symbol RXRG [Human]
Official name retinoid X receptor, gamma
Species Human (Homo sapiens)
Entrez Gene ID 6258
Transcript(s) for this gene NM_001009598 (633 bp), NM_001256570 (2263 bp), NM_001256571 (1680 bp), NM_006917 (2205 bp), NR_033824 (803 bp), ENST00000465764, ENST00000359842, ENST00000619224
Official symbol Rxrg [Mouse]
Official name retinoid X receptor gamma
Species Mouse (Mus musculus)
Entrez Gene ID 20183
Transcript(s) for this gene NM_001159731 (1695 bp), NM_009107 (2151 bp)
Official symbol Rxrg [Rat]
Official name retinoid X receptor gamma
Species Rat (Rattus norvegicus)
Entrez Gene ID 83574
Transcript(s) for this gene NM_031765 (1990 bp), XM_341151 (1735 bp), XM_006250212 (2121 bp)
Official symbol Rxrb [Mouse]
Official name retinoid X receptor beta
Species Mouse (Mus musculus)
Entrez Gene ID 20182
Transcript(s) for this gene NM_001205214 (2633 bp), NM_001205215 (2356 bp), NM_001205216 (2344 bp), NM_011306 (2621 bp), ENSMUST00000174740
Official symbol RXRB [Human]
Official name retinoid X receptor, beta
Species Human (Homo sapiens)
Entrez Gene ID 6257
Transcript(s) for this gene NM_021976 (2919 bp), NM_001270401 (2931 bp), ENST00000483821, NM_001291989 (2212 bp), XM_005249278 (2606 bp), XM_005249279 (1356 bp), XM_005272854 (2606 bp), XM_005272855 (1356 bp), XM_005275007 (2606 bp), XM_005275008 (1356 bp), XM_005275149 (2607 bp), XM_005275150 (1356 bp), XM_005275276 (2606 bp), XM_005275277 (1356 bp), XM_005275438 (2607 bp), XM_005275439 (1356 bp), XM_006715162 (2537 bp), XM_006725494 (2536 bp), XM_006725708 (2537 bp), XM_006725825 (2537 bp), XM_006725917 (2536 bp), XM_006726010 (2537 bp), ENST00000374680, ENST00000374685
Official symbol Rxrb [Rat]
Official name retinoid X receptor beta
Species Rat (Rattus norvegicus)
Entrez Gene ID 361801
Transcript(s) for this gene NM_206849 (2481 bp), XM_342095 (1486 bp), XM_006255960 (3003 bp), XM_006255961 (2496 bp)