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

beta -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 thes e promoters is thought to be stable under most circumstances. To determine if expression of beta -galactosidase from the commonly used beta -galactosi dase 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 RSV beta- or CMV beta- or pTK beta plasmids alone or together with a reporter construct in which lucifer ase transcription is driven by the c-fos promoter. Luciferase and/or beta - galactosidase activity was measured from the lysate of cells treated with a ngiotensin If or serum. We found that agonists increased the transcriptiona l activity of the different beta -galactosidase plasmids. The effect of ang iotensin II and serum was different on the different promoters. Finally, co transfection of other plasmids also modulated beta -galactosidase activity. These agonist induced variations of beta -galactosidase activity may influ ence the analysis and interpretation of the results in a systematic manner. Consequently we conclude that the use of a second reporter system to contr ol for transfection efficiency in certain types of experiments may lead to a systematic error and is questionable as a general procedure. (C) 2001 Els evier Science B.V. All rights reserved.