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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 = 213.5
Average Z' factor at maximum response rate = 0.77
Average coefficient of variation (CV%) = 7.6%
Excellent signal to noise ratio and dose dependent
response
Cignal CRE reporter assay showed dose dependent
increase in the activity of cAMP pathway: 293-H cells were
transfected with CRE reporter and positive control (for transfection
protocol refer our user manual). After 16 hours of transfection, cells
were treated with different doses of forskolin for 6 hours. (Forskolin
increases the intracellular level of cAMP). 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. Cignal CRE
reporter assay measured 213 fold increase in the transcription activity of
CRE-binding protein (CREB) and, in turn, in the activity of corresponding
cAMP signaling pathway by 10µM forskolin.
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