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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.
General performance
Average maximum response rate = 280
Average Z' factor at maximum response rate = 0.84
Average coefficient of variation (CV%) = 7.6%
Excellent signal to noise ratio
Cignal RBP-Jk reporter assay showed upregulation of Notch signaling activity: 293-H cells were transfected with RBP-Jk reporter, negative control and positive control (for transfection protocol refer our user manual). After 16 hours of transfection medium was changed to complete medium. After 24 hours of transfection, cells were infected with 100 MOI of recombinant adenoviruses expressing constitutive active Notch1 (Ad-NICD) or 100 MOI of recombinant adenovirus expressing GFP (Ad-GFP). Dual Luciferase assay was performed 18 hours after infection, 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. Cignal RBP-Jk reporter assay measured ~600 fold increase in the RBP-JK transcription activity and, in turn, in the corresponding Notch signaling pathway by overexpression of constitutive active Notch1.
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