Is it acceptable to correct for transfection efficiency, when determining the level of gene expression knock down by an shRNA expression vector?
You should calculate the transfection efficiency, and use this as a criteria for deciding whether or not to proceed with qRT-PCR analyses. Expression knock down validation studies should only be carried out if the transfection efficiency is 80% or higher. However, we strongly discourage applying a transfection efficiency correction factor to qRT-PCR generated gene expression knock down data. We also strongly discourage applying a transfection efficiency correction factor to any functional assay results. The observed level of knock down in a mixed population of transfected and untransfected cells may fall below the confidence interval for qPCR, and correcting for transfection efficiency would then artificially inflate the real level of expression knock down and/or change in functional activity.
