Toxicological housekeeping genes: Do they really keep the house?

It is assumed that the expression of housekeeping genes is constant regardless of experimental conditions. In toxicology, this assumption has indeed become a misconception of reasonable concern, as these so-called housekeeping genes vary considerably across different experimental conditions and thereby lead to an erroneous interpretation of the expression profile of a target gene. Given that the choice of reference gene will ultimately influence statistical interpretation of toxicological data, it is essential to validate potential reference genes prior to their use, to establish their suitability for a specific experimental purpose. Therefore, the aim of this study is to quantitatively evaluate the most commonly used housekeeping genes in toxicology research for their suitability as reference endpoints, and thus provide toxicology researchers who have little experience in molecular biology but find themselves interested or involved with gene expression analysis with a summary of information necessary for re-evaluating their procedures. We show that the expression pattern of beta-actin, beta-tubulin, 18S ribosomal RNA (18S rRNA), and elongation factor-1 alpha (EF-1 alpha), representing commonly used housekeeping genes in toxicology, was modulated on the basis of random exposure condition and time, in both in vivo and in vitro test systems of Atlantic salmon (Salmo salar). Based on the data presented herein and several other reports by other researchers, there are very few biological justifications to refer to anything as a housekeeping gene in real-time PCR assays for toxicological research. However, given the absolute need for normalization genes to correct for sample-to-sample variations, the choice of internal control gene should be determined empirically on the basis of the individual exposure condition and by the individual researcher.