Reference Gene Selection for Expression Analysis of Hepatic Genes Responding to Fasting via Transcriptomic Analysis

Abstract

The liver of mammals is a critical organ that houses numerous physiological processes, including macronutrient metabolism. Regarding nutrient metabolism, the liver shows a quick response to nutrient status fluctuation, such as feeding and fasting. Indeed, fasting is a powerful tool to uncover the molecular mechanism through which the expression of hepatic genes is regulated. The most widely applied approach to test gene expression at the RNA level is relative quantitative polymerase chain reaction (qPCR) using stably expressed genes as an internal reference. In this study, we leveraged transcriptomic analysis to screen the hepatic genes whose expression levels were stable in response to fasting. Our data suggested that expression levels of several well-known reference genes such as beta-actin and Eef1a1 are dramatically altered upon fasting. Further assessment indicated that Itm2b is the most stably expressed hepatic gene showing no alteration to fasting. Compared to beta-actin and Eef1a1, similar results and conclusions as transcriptomic analysis were obtained using Itm2b as an internal reference in qPCR when we assessed the effect of fasting on the expression of genes involved in peroxisomal fatty acid oxidation, fatty acid synthesis, and gluconeogenesis.