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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.
Excellent signal to noise ratio
293-H cells were transfected with FOXO-reporter and positive
control (for transfection protocol, please refer to the user manual). After 16
hours of transfection medium was changed to growth medium. After 24 hours of
transfection, cells were infected with 10 MOI of recombinant adenoviruses
expressing FOXO3A (Ad-FOXO3A) or 10 MOI of recombinant
adenovirus expressing GFP (Ad-GFP). Dual Luciferase assay was performed 42 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.
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