The importance of and a method for including transfection efficiency into real‐time PCR data analyses

The polymerase chain reaction (PCR) is widely used to ascertain absolute or relative changes in the expression levels of specific genes as a function of cell type or in response to changes in environmental stimuli. Real-time PCR is an advance which allows for the analysis of gene expression over a wide range of initial cDNA concentrations, where the cDNA is the product of reverse transcriptase reactions applied to RNA samples. With the advent and advances in gene delivery technologies, it is now common for the cellular responses under scrutiny to be initiated via the expression of an exogenously delivered gene. When transfection (or transduction) is a part of the procedure used to prepare cell samples for real-time PCR, it is necessary to take the efficiency of gene delivery into account. Here a robust mathematical model for such analyses is derived, and validated with theoretical and experimental support. Comparison to existing analysis methods is presented to demonstrate the high significance of noting transfection, loading, and primer PCR efficiencies when processing PCR data.