Activation of pRL-TK by 12S E1A oncoprotein: drawbacks of using an internal reference reporter in transcription assays.

In transient expression assays for transcription, a vector carrying the experimental reporter is usually co-transfected with a second vector containing a distinct reporter gene as a control. The second reporter is linked to a constitutive promoter driving a low-level transcription that is unresponsive to the experimental trans-acting transcription factors used. The use of dual reporters enables the normalization of the experimental gene transcription with respect to the control reporter transcription. This method is expected to minimize the inherent variability in transfection data caused by changes in cell density and viability, cell lysis, and the recovery of samples at various stages of the experiment. Here, we report that one of the most widely used internal control reporters, the Renilla luciferase plasmid (pRL-TK), is unresponsive to human tumor suppressor protein p53, a potent transcriptional regulator; however, the reporter transcription is enhanced by another well-known transcriptional regulator, the adenoviral 125 EIA oncoprotein, thereby disqualifying pRL-TK as a universal internal control reporter for transcription assays. Our results reveal the necessity of stringent experiments to test the responsiveness of internal control plasmids to transcriptional regulators present in the assay to avoid the misinterpretation of transcriptional analysis data.