Identification of optimal reference genes for RT-qPCR in the rat hypothalamus and intestine for the study of obesity.

BACKGROUND: Obesity has a complicated metabolic pathology, and defining the underlying mechanisms of obesity requires integrative studies with molecular end points. Real-time quantitative PCR (RT-qPCR) is a powerful tool that has been widely utilized. However, the importance of using carefully validated reference genes in RT-qPCR seems to have been overlooked in obesity-related research. The objective of this study was to select a set of reference genes with stable expressions to be used for RT-qPCR normalization in rats under fasted vs re-fed and chow vs high-fat diet (HFD) conditions. DESIGN: Male long-Evans rats were treated under four conditions: chow/fasted, chow/re-fed, HFD/fasted and HFD/re-fed. Expression stabilities of 13 candidate reference genes were evaluated in the rat hypothalamus, duodenum, jejunum and ileum using the ReFinder software program. The optimal number of reference genes needed for RT-qPCR analyses was determined using geNorm. RESULTS: Using geNorm analysis, we found that it was sufficient to use the two most stably expressed genes as references in RT-qPCR analyses for each tissue under specific experimental conditions. B2M and RPLP0 in the hypothalamus, RPS18 and HMBS in the duodenum, RPLP2 and RPLP0 in the jejunum and RPS18 and YWHAZ in the ileum were the most suitable pairs for a normalization study when the four aforementioned experimental conditions were considered. CONCLUSIONS: Our study demonstrates that gene expression levels of reference genes commonly used in obesity-related studies, such as ACTB or RPS18, are altered by changes in acute or chronic energy status. These findings underline the importance of using reference genes that are stable in expression across experimental conditions when studying the rat hypothalamus and intestine, because these tissues have an integral role in the regulation of energy homeostasis. It is our hope that this study will raise awareness among obesity researchers on the essential need for reference gene validation in gene expression studies.