The use of a second reporter plasmid as an internal standard to normalize luciferase activity in transient transfection experiments may lead to a systematic error

Abstract

β-galactosidase reporter plasmids containing different viral or minimal promoters are commonly used to correct variable transfection efficiencies in transient transfection experiments. The transcriptional activity of these promoters is thought to be stable under most circumstances. To determine if expression of β-galactosidase from the commonly used β-galactosidase plasmids remains stable upon stimulation of the cells with agonists we performed transient transfection experiments. CHO cells stably expressing the rat AT 1A receptor were transfected with RSVβ- or CMVβ- or pTKβ plasmids alone or together with a reporter construct in which luciferase transcription is driven by the c-fos promoter. Luciferase and/or β-galactosidase activity was measured from the lysate of cells treated with angiotensin II or serum. We found that agonists increased the transcriptional activity of the different β-galactosidase plasmids. The effect of angiotensin II and serum was different on the different promoters. Finally, cotransfection of other plasmids also modulated β-galactosidase activity. These agonist induced variations of β-galactosidase activity may influence the analysis and interpretation of the results in a systematic manner. Consequently we conclude that the use of a second reporter system to control for transfection efficiency in certain types of experiments may lead to a systematic error and is questionable as a general procedure.