Selection of reference gene for qPCR analysis of hypothalamic appetite regulation

Abstract

Identification of an internal control gene for normalization is required for qRT‐PCR experiments. Vandersompele et al (2002) have demonstrated the need for housekeeper genes that are unaffected by experimental treatments. Several investigators have selected β‐actin as the preferred housekeeper in examining the effects of sugars on hypothalamic appetite regulatory genes. We set out to determine if access to different sugar solutions could alter hypothalamic gene expression in rats. Presented are three sets of observations related to the selection of a housekeeper. First β‐actin varied across treatments, making it a poor choice. Second, a poorly selected housekeeper can result in reporting of false conclusions. When fold change was calculated using β‐actin, differences in AGRP were observed among treatment groups. However, when analyzed with RPLP as housekeeper no differences were observed. Third, sometimes using a poor housekeeper does not lead to false conclusions. Differences in some genes were significant independent of the housekeeper. Here we demonstrate that housekeeper selection can impact conclusions. Supported in part by a grant from the Maryland Agricultural Experiment Station.