Control selection for RNA quantitation

The study of mammalian gene expression is often carried out at the level of mRNA. In such analyses, one usually measures the amount of an mRNA of inte rest under different conditions such as stress, growth, development, cell a nd tissue localization or as part of an evaluation of the effects of gene t ransfection. A variety of techniques exist to measure gene expression and m ost commonly involve Northern hybridization analysis, ribonuclease protecti on or RT-PCR. Common to all of these assays is the inclusion of a so-called loading or internal control (i.e., analysis of an mRNA that does not chang e in relative abundance during the course of treatments). Here, we discuss the uses and pitfalls of the most popular of these controls, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and beta-actin, with special emphasis on precautions associated with the use of GAPDH.