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Cignal Finder Reporter Arrays

For discovery of signaling pathway response to gene modification or chemical treatment

  • Analysis of multiple pathways in a single experiment
  • Flexible formats
  • Assays gene knockdown and overexpression
  • Assays chemical modulators

Cignal Finder Reporter Array allows for the comprehensive analysis of 10 signaling pathways. By screening pathway activities simultaneously, relevant pathways are quickly identified for further analysis. Cignal Finder Reporter Arrays pinpoint the pathways perturbed by a specific gene or drug.

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


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Signaling pathways affected by p53 knockdown.

HEK-293H cells were cotransfected with either p53 siRNA or a negative control siRNA, in combination with each reporter assay and the negative control from the Cancer 10-Pathway Reporter Array plate. 16 hours after transfection, medium was changed to complete medium. 48 hours after transfection, the dual-luciferase assay was performed. Results are expressed as fold change.

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Signaling pathways affected by TNFα.

HeLa cells were reverse transfected with the Immune Response 10-Pathway Cignal Finder Reporter Array. 16 hours after transfection, medium was changed to assay medium. 32 hours after transfection, cells were treated with 5 ng/ml TNFα or left untreated. After 6 hours treatment, dual-luciferase assays were performed. Results are expressed as fold change.

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Cignal Finder Array, tube format procedure.
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Cignal Finder Array, plate format procedure.
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Identification of optimal drug dose.

HepG2 cells were reverse transfected with each reporter assay and the controls on the Stress and Toxicity 10-Pathway Reporter Array plate. 16 hours after transfection, the medium was changed to complete medium. After 30 hours, cells were treated with increasing dosages of tunicamycin (0–4 µg/ml). After 48 hours, the dual-luciferase assay was performed; results are expressed as fold change. 0.125 µg/ml Tunicamycin is the lowest effective non-toxic dose for glycoprotein synthesis inhibition.

Performance

Cignal Finder Reporter Arrays can be used in many applications.

Identification of optimal drug doses

Toxicity is one of the primary reasons potential drug candidates fail during development. Identifying potential toxicity and the cellular response to a given concentration or compound can benefit future studies. Toxic compounds or dosages activate various stress response pathways. Cell-based luciferase reporter assays were used to measure 10 stress response pathways using a single 96-well plate.

Tunicamycin inhibits the enzyme GlcNAc phosphotransferase (GPT) and induces endoplasmic reticulum (ER) stress. In this study, the Stress and Toxicity 10-Pathway Reporter Array was used to determine the lowest effective concentration of Tunicamycin without activating cellular stress pathways. Results showed that 0.125 µg/ml Tunicamycin is the lowest effective non-toxic dose for glycoprotein synthesis inhibition (see figure "Identification of optimal drug dose").

Cell response to loss of a cancer signaling pathway

The most well-studied tumor suppressor gene is p53. To understand more about the biological function of p53, it is important to measure the signaling pathways affected by p53 knockdown. The Cancer 10-Pathway Reporter Array can help to identify key cancer signaling pathways modulated by p53 knockdown (see figure "Signaling pathways affected by p53 knockdown").

Results showed that knockdown of p53 gene expression downregulates p53 signaling, while upregulating Notch, hypoxia, and MAPK/ERK signaling in HEK-293H cells. Notch signaling is known to be frequently deregulated in human malignancies. Activation of Notch signaling by p53 RNA interference suggests that Notch may function as a proto-oncogene.

Measurement of signaling in response to cytokines

Tumor necrosis factor alfa (TNFα) is a pleiotropic inflammatory cytokine. It is important to determine the key immunology signaling pathways modulated by TNFα. The Immune Response 10-Pathway Cignal Finder Reporter Array may provide valuable information about the signaling pathways involved in the biological response to TNFα.

The Immune Response 10-Pathway Cignal Finder Reporter Array reveals that TNFα activates the NFkB and MAPK/JNK signaling pathways in HeLa cells (see figure Signaling pathways affected by TNFalpha).

Principle

Each array includes 10 Cignal Reporter Assays and 2 controls in either a transfection-ready tube or plate format.
All reporter assays are based on dual-luciferase technology. Each reporter consists of a mixture of a pathway-focused transcription factor-responsive firefly luciferase construct and a constitutively expressing Renilla luciferase construct.

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 serves 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.

Procedure

The procedure is comprised of 3 simple steps:

Transfect Cignal Finder Reporter Assays and test nucleic acids into cells 
Treat with siRNA, protein, peptide, or small molecule of interest 
Perform reporter quantitation using luciferase activity assays
Cignal Finder Reporter Array: Tube Format

Cignal Finder Reporter Arrays in tube format are delivered in 12-tube strips, along with positive and negative controls. The assays are immediately ready for use in the transfection or reverse transfection of the reporter assays into the cell lines of interest (see flowchart "Cignal Finder Array, tube format procedure").

Cignal Finder Reporter Array: Plate Format

Cignal Finder Reporter Arrays in plate format are delivered in a 96-well cell culture plate. Each reporter and control assay is dried down in each column of the plate (8 wells per assay; see flowchart "Cignal Finder Array, plate format procedure").

Applications

Cignal Reporter Arrays are highly suited for research into phenotypes associated with RNAi or overexpression experiments, biological responses to small molecules or compounds, and mechanisms of action of proteins, peptides, and ligands.

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Brochures & Guides (1)
For cell-based analysis of pathway signaling activity
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Kit Handbooks (3)
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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Stress & Toxicity 10-Pathway Reporter Array Cignal Finder Reporter Array
Introduction
The Stress & Toxicity Reporter Array analyzes the activity of ten pathways associated with cytotoxicity and cellular stress. Each pathway reporter is a mixture of a pathway-responsive luciferase construct and a constitutively expressed Renilla luciferase construct. The pathway-responsive constructs encode the firefly luciferase reporter gene under the control of a specific transcription factor associated with a pathway. Using a simple dual-luciferase assay, the activity of each pathway is measured to determine the effect of various treatments, such as gene knockdown, over-expression, and chemical compounds.
Gene List

Tube number  Pathway  Cignal Reporter Assay
 1  Oxidative stress   Nrf2/Nrf1 Cignal Reporter Assay
 2  DNA damage   p53 Cignal Reporter Assay
 3  NFκB   NFκB Cignal Reporter Assay
 4  Hypoxia   HIF1 Cignal Reporter Assay
 5  ER stress   CBF/NF-Y/YY1 Cignal Reporter Assay
 6  Heavy metal stress   MTF1 Cignal Reporter Assay
 7  Heat shock  HSF1 Cignal Reporter Assay
 8  Glucocorticoid   Glucocorticoid Receptor Cignal Reporter Assay
 9  JNK   AP1 Cignal Reporter Assay
 10  Xenobiotic   AhR Cignal Reporter Assay
 11  Negative Control Cignal Reporter Assay
 12  Positive Control Cignal Reporter Assay
Name Cignal Finder Stress & Toxicity 10-Pathway Reporter Array (tube format) (CCA-007L)