Housekeeping genes revisited: Different expressions depending on gender, brain area and stressor

Housekeeping gene (HKG) mRNAs are used to normalize expression data of genes of interest in quantitative reverse transcriptase polymerase chain reaction studies. Such normalization assumes constant HKG gene expression under all circumstances. Although sporadic evidence suggests that HKG expression may not always fulfill this requirement and, therefore, such normalization may lead readily to erroneous results, this fact is generally not sufficiently appreciated by investigators. Here, we have systematically analyzed the expression of three common HKGs, glyceraldehyde-3-phosphate dehydrogenase, ribosomal subunit 18S and β-actin, in two different stress paradigms, in various brain areas, in male and in female rats. HKG expressions differed considerably with respect to brain area, type of stressor and gender, in an HKG-specific manner. Therefore, we conclude that before final experimentation, pilot expression studies are necessary to select an HKG which expression is unaffected by the experimental factor(s), allowing reliable interpretation of expression data of genes of interest.