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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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Transfection protocols for specific cell types and plate formats that save you the time and effort of adapting existing protocols to fit your requirements. Simply select the cell type, nucleic acid, and culture format to receive a QIAGEN transfection protocol to print out or download in convenient PDF format.
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EGR1 Reporter Assay Kit (GFP) Cignal Reporter Assay Kits
Introduction
The Cignal EGR1 Reporter Assay Kit is designed to measure the transcriptional activity of the early growth response 1 (EGR1) transcription factor, which belongs to the EGR family of C2H2-type zinc-finger proteins. The target genes of EGR1, a nuclear protein, are required for differentiation and mitogenesis. The kit contains transfection-ready EGR1 reporter as well as positive and negative controls. The EGR1 reporter encodes the Monster GFP gene under the control of a minimal (m)CMV promoter and tandem repeats of the EGR1 transcriptional response element (TRE). We have experimentally optimized the number of response elements as well as the intervening sequence between response elements to maximize the signal to noise ratio.
CCS-3021G

The Cignal EGR1 Reporter Assay detects EGR1 transcription activity. 293-H cells were transfected with the Cignal EGR1 Reporter, negative control, and positive control (for the transfection protocol, refer to the Cignal Reporter Assay Handbook). After 24 hours of transfection, medium was changed to assay medium (DMEM + 0.5% FBS + 0.1 mM NEAA). After 26 hours of transfection, cells were [A] and [C] untreated, or [B] and [D] treated with PMA (10 ng/ml). After 24 hours of treatment, bright field and fluorescent images were taken of the cultures transfected with the Cignal EGR1-GFP Reporter.
Name Cignal EGR1 Reporter Assay Kit (GFP) (CCS-3021G)
Official symbol HPSE [Human]
Official name heparanase
Species Human (Homo sapiens)
Entrez Gene ID 10855
Transcript(s) for this gene NM_001098540 (4630 bp), NM_001166498 (4446 bp), NM_001199830 (4388 bp), NM_006665 (4668 bp), ENST00000311412, ENST00000405413, ENST00000512196, ENST00000513463
Official symbol Hpse [Mouse]
Official name heparanase
Species Mouse (Mus musculus)
Entrez Gene ID 15442
Transcript(s) for this gene NM_152803 (3082 bp), XM_982223 (2043 bp)
Official symbol MAOB [Human]
Official name monoamine oxidase B
Species Human (Homo sapiens)
Entrez Gene ID 4129
Transcript(s) for this gene NM_000898 (2611 bp), ENST00000487544, XM_005272607 (2596 bp), XM_005272608 (2556 bp), ENST00000378069
Official symbol Maob [Mouse]
Official name monoamine oxidase B
Species Mouse (Mus musculus)
Entrez Gene ID 109731
Transcript(s) for this gene NM_172778 (2428 bp)
Official symbol Maob [Rat]
Official name monoamine oxidase B
Species Rat (Rattus norvegicus)
Entrez Gene ID 25750
Transcript(s) for this gene NM_013198 (2389 bp)
Official symbol VEGFA [Human]
Official name vascular endothelial growth factor A
Species Human (Homo sapiens)
Entrez Gene ID 7422
Transcript(s) for this gene NM_001025366 (3677 bp), NM_001025367 (3608 bp), NM_001025368 (3554 bp), NM_001025369 (3519 bp), NM_001025370 (3422 bp), NM_001033756 (3488 bp), NM_001171622 (3392 bp), NM_001171623 (3677 bp), NM_001171624 (3626 bp), NM_001171625 (3608 bp), NM_001171626 (3554 bp), NM_001171627 (3519 bp), NM_001171628 (3422 bp), NM_001171629 (3488 bp), NM_001171630 (3392 bp), NM_001204384 (3494 bp), NM_001204385 (3494 bp), NM_003376 (3626 bp), ENST00000518538, NM_001287044 (2569 bp), ENST00000230480, ENST00000324450, ENST00000372055, ENST00000372064, ENST00000372067, ENST00000372077, ENST00000413642, ENST00000417285, ENST00000425836, ENST00000457104, ENST00000482630, ENST00000518689, ENST00000518824, ENST00000520948, ENST00000523125, ENST00000523873, ENST00000523950, ENST00000611736, ENST00000615393, ENST00000617771, ENST00000621747
Official symbol Vegfa [Mouse]
Official name vascular endothelial growth factor A
Species Mouse (Mus musculus)
Entrez Gene ID 22339
Transcript(s) for this gene NM_001025250 (3547 bp), NM_001025257 (3343 bp), NM_001110266 (2857 bp), NM_001110267 (2785 bp), NM_001110268 (2653 bp), NM_009505 (3475 bp), ENSMUST00000150327, NM_001287056 (3547 bp), NM_001287057 (3475 bp), NM_001287058 (3343 bp), NM_001317041 (3475 bp)
Official symbol Vegfa [Rat]
Official name vascular endothelial growth factor A
Species Rat (Rattus norvegicus)
Entrez Gene ID 83785
Transcript(s) for this gene NM_001110333 (3474 bp), NM_001110334 (3342 bp), NM_001110335 (2616 bp), NM_001110336 (2544 bp), NM_031836 (3546 bp), NM_001287107 (3546 bp), NM_001287108 (3474 bp), NM_001287110 (3342 bp), NM_001287111 (3414 bp), NM_001287112 (3414 bp), NM_001287113 (3425 bp), NM_001287114 (3293 bp), NR_105011 (0 bp)
Official symbol CGA [Human]
Official name glycoprotein hormones, alpha polypeptide
Species Human (Homo sapiens)
Entrez Gene ID 1081
Transcript(s) for this gene NM_000735 (768 bp), NM_001252383 (861 bp), ENST00000369582, ENST00000610310, ENST00000627148, ENST00000630630
Official symbol Cga [Mouse]
Official name glycoprotein hormones, alpha subunit
Species Mouse (Mus musculus)
Entrez Gene ID 12640
Transcript(s) for this gene NM_009889 (691 bp), XM_006537586 (687 bp)
Official symbol Cga [Rat]
Official name glycoprotein hormones, alpha polypeptide
Species Rat (Rattus norvegicus)
Entrez Gene ID 116700
Transcript(s) for this gene NM_053918 (667 bp)
Official symbol IL1B [Human]
Official name interleukin 1, beta
Species Human (Homo sapiens)
Entrez Gene ID 3553
Transcript(s) for this gene NM_000576 (1498 bp), ENST00000496280, ENST00000491056, XM_006712496 (1692 bp), ENST00000263341
Official symbol Il1b [Mouse]
Official name interleukin 1 beta
Species Mouse (Mus musculus)
Entrez Gene ID 16176
Transcript(s) for this gene NM_008361 (1348 bp), ENSMUST00000155994, XM_006498795 (2649 bp)
Official symbol Il1b [Rat]
Official name interleukin 1 beta
Species Rat (Rattus norvegicus)
Entrez Gene ID 24494
Transcript(s) for this gene NM_031512 (1339 bp)
Official symbol NAB2 [Human]
Official name NGFI-A binding protein 2
Species Human (Homo sapiens)
Entrez Gene ID 4665
Transcript(s) for this gene NM_005967 (2725 bp), ENST00000555857, XM_005268894 (2519 bp), ENST00000300131, ENST00000342556
Official symbol Nab2 [Mouse]
Official name Ngfi-A binding protein 2
Species Mouse (Mus musculus)
Entrez Gene ID 17937
Transcript(s) for this gene NM_001122895 (2401 bp), NM_008668 (2593 bp), XM_006513330 (2912 bp), XM_006513331 (2720 bp)
Official symbol Nab2 [Rat]
Official name Ngfi-A binding protein 2
Species Rat (Rattus norvegicus)
Entrez Gene ID 314910
Transcript(s) for this gene NM_001134874 (3366 bp), XM_001056620 (3506 bp), XM_235224 (3498 bp), XM_006241459 (3814 bp)
Official symbol Sp1 [Mouse]
Official name trans-acting transcription factor 1
Species Mouse (Mus musculus)
Entrez Gene ID 20683
Transcript(s) for this gene NM_013672 (7818 bp), ENSMUST00000170042, XM_006520673 (7851 bp), XM_006520674 (7713 bp), XM_011245533 (7864 bp)
Official symbol SP1 [Human]
Official name Sp1 transcription factor
Species Human (Homo sapiens)
Entrez Gene ID 6667
Transcript(s) for this gene NM_001251825 (7523 bp), NM_003109 (7614 bp), NM_138473 (7667 bp), XM_006719570 (7905 bp), XM_006719571 (7761 bp), ENST00000327443, ENST00000426431
Official symbol Sp1 [Rat]
Official name Sp1 transcription factor
Species Rat (Rattus norvegicus)
Entrez Gene ID 24790
Transcript(s) for this gene NM_012655 (7718 bp)
Official symbol Tnf [Mouse]
Official name tumor necrosis factor
Species Mouse (Mus musculus)
Entrez Gene ID 21926
Transcript(s) for this gene NM_013693 (1653 bp), NM_001278601 (1605 bp)
Official symbol TNF [Human]
Official name tumor necrosis factor
Species Human (Homo sapiens)
Entrez Gene ID 7124
Transcript(s) for this gene NM_000594 (1686 bp), ENST00000449264
Official symbol Tnf [Rat]
Official name tumor necrosis factor
Species Rat (Rattus norvegicus)
Entrez Gene ID 24835
Transcript(s) for this gene NM_012675 (1687 bp)
Official symbol Birc5 [Mouse]
Official name baculoviral IAP repeat-containing 5
Species Mouse (Mus musculus)
Entrez Gene ID 11799
Transcript(s) for this gene NM_001012272 (867 bp), NM_001012273 (3416 bp), NM_009689 (977 bp)
Official symbol BIRC5 [Human]
Official name baculoviral IAP repeat containing 5
Species Human (Homo sapiens)
Entrez Gene ID 332
Transcript(s) for this gene NM_001012270 (2537 bp), NM_001012271 (2724 bp), NM_001168 (2655 bp), ENST00000592115, ENST00000301633, ENST00000350051, ENST00000374948, ENST00000590449, ENST00000592734
Official symbol Hpse [Rat]
Official name heparanase
Species Rat (Rattus norvegicus)
Entrez Gene ID 64537
Transcript(s) for this gene NM_022605 (3177 bp)
Official symbol Ace [Rat]
Official name angiotensin I converting enzyme
Species Rat (Rattus norvegicus)
Entrez Gene ID 24310
Transcript(s) for this gene NM_012544 (4142 bp)
Official symbol ACE [Human]
Official name angiotensin I converting enzyme
Species Human (Homo sapiens)
Entrez Gene ID 1636
Transcript(s) for this gene NM_000789 (4969 bp), NM_001178057 (3141 bp), NM_152830 (3264 bp), NM_152831 (4863 bp), ENST00000584865, XM_005257110 (4875 bp), XM_006721737 (3132 bp), ENST00000290863, ENST00000290866, ENST00000413513, ENST00000428043
Official symbol Ace [Mouse]
Official name angiotensin I converting enzyme (peptidyl-dipeptidase A) 1
Species Mouse (Mus musculus)
Entrez Gene ID 11421
Transcript(s) for this gene NM_009598 (3129 bp), NM_207624 (4906 bp), XM_112531 (4363 bp), ENSMUST00000151657, NM_001281819 (5091 bp)
Official symbol Egr1 [Rat]
Official name early growth response 1
Species Rat (Rattus norvegicus)
Entrez Gene ID 24330
Transcript(s) for this gene NM_012551 (1527 bp)
Official symbol EGR1 [Human]
Official name early growth response 1
Species Human (Homo sapiens)
Entrez Gene ID 1958
Transcript(s) for this gene NM_001964 (3136 bp), ENST00000239938
Official symbol Egr1 [Mouse]
Official name early growth response 1
Species Mouse (Mus musculus)
Entrez Gene ID 13653
Transcript(s) for this gene NM_007913 (3072 bp)
Official symbol Egfr [Mouse]
Official name epidermal growth factor receptor
Species Mouse (Mus musculus)
Entrez Gene ID 13649
Transcript(s) for this gene NM_007912 (2678 bp), NM_207655 (5983 bp), ENSMUST00000139722, XM_006514491 (6041 bp)
Official symbol EGFR [Human]
Official name epidermal growth factor receptor
Species Human (Homo sapiens)
Entrez Gene ID 1956
Transcript(s) for this gene NM_005228 (5616 bp), NM_201282 (2239 bp), NM_201283 (1595 bp), NM_201284 (2865 bp), ENST00000485503, ENST00000275493, ENST00000342916, ENST00000344576, ENST00000420316, ENST00000442591, ENST00000454757, ENST00000455089
Official symbol Egfr [Rat]
Official name epidermal growth factor receptor
Species Rat (Rattus norvegicus)
Entrez Gene ID 24329
Transcript(s) for this gene NM_031507 (4161 bp), XM_008770416 (9579 bp), XM_008770417 (9537 bp), XM_008770418 (3800 bp)
Official symbol Birc5 [Rat]
Official name baculoviral IAP repeat-containing 5
Species Rat (Rattus norvegicus)
Entrez Gene ID 64041
Transcript(s) for this gene NM_022274 (903 bp)

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