| |
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 = 42.2
Average Z' factor at maximum response rate = 0.77
Average coefficient of variation (CV%) = 6.7%
Excellent signal to noise ratio
Cignal SRE reporter assay can measure upregulation of serum response factor (SRF) transcription activity: 293-H cells were transfected with SRE reporter, negative control and positive control (for transfection protocol refer our user manual). After 16 hours of transfection, medium was changed to assay medium (Opti-MEM + 0.5% FBS + 0.1mM NEAA + 1mM Sodium pyruvate + 100 U/ml penicillin + 100 µg/ml streptomycin). After 40 hours of transfection, cells were treated with 10% serum, and 10% serum and 100 ng/ml EGF 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 done in triplicates, and the standard deviation is indicated.
Back to Top
|
|
