BACKGROUND Obesity has a complicated metabolic pathology, and defining the underlying mechanisms of obesity requires integrative studies with molecular endpoints. 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 be 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 the13 candidate reference genes were evaluated in the rat hypothalamus, duodenum, jejunum, and ileum using 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. Unique subsets of reference genes out of the 13 candidate genes were identified, each of which is specific for one type of rat tissue (hypothalamus, duodenum, jejunum, or ileum) under a different combination of diet and feeding condition. 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 play an integral role in 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.
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