Figure 3

Importance of proper normalization for luciferase assays. (a) Assessment of basal activity of the RL vectors that are commonly used in luciferase reporter assays in Drosophila clone 8 cells in 96-well plate format. The SV40-RL, TK-RL, and Copia-RL vectors display no activity or very weak basal activity; approximately two to four times above background or negative control (no RL reporter added). CMV-RL displays weak activity (approximately 12 times above background) whereas pIZT-RL and pAct-RL display moderate basal activity (approximately 20 to 30 times above background). PolIII-RL displays the most robust activity among all the RL vectors tested (>1,000 times above background). (b) pAct-RL can be activated by transfecting cDNA expressing Wg or by dsRNA-mediated knockdown of known negative regulators (GSK-3β, APC) of the Wg pathway in S2R+ cells, thus rendering it unusable as a control for transfection efficiency and cell viability in luciferase assays. (c) RL counts produced by transfection of the indicated Renilla control reporter and treatment with the indicated dsRNA in the Hh signaling assay. (d) Firefly luciferase counts produced by the ptcΔ136 reporter when cotransfected with the indicated Renilla control reporter and dsRNA. (e) Graph showing the fold difference in ptcΔ136 reporter activity in clone 8 cells treated with smo dsRNA versus GFP dsRNA in the presence of the indicated Renilla control reporter. Bars are the ratio of GFP dsRNA treated: smo dsRNA treated taken from the data in (c) and (d). All luciferase reporter assays were performed in triplicate and error bars represent the standard error between the three data points.