Variation in enzymatic transient gene expression assays

We examined causes for high variability in data from enzymatic transient gene expression assays. Our results strongly suggest that variation in transfection efficiency is the major cause of data variation and can seriously compromise valid interpretation of data. We compared averaging data from multiple transfections and cotransfection of a second reporter gene as methods for correcting for variation in transfection efficiency. We found that transfection efficiency can be so highly variable that neither method necessarily overcomes the resulting bias in data. Depending upon the degree in variation in transfection efficiency, a combination of the two methods may be advisable. The need to normalize data for transfection efficiency is dependent upon the difference in strengths of promoters being tested and the relative variability of the transfection method used. We also show that the level of reporter gene expression between transfection experiments performed on different days can vary by more than 10-fold.