Identification of Suitable Reference Genes for Gene Expression Studies in Tissues from Fructose-Fed Rats

Abstract

Selection of appropriate endogenous control for normalization in a specific experimental paradigm is a critical step in the quantification of gene expression. The aim of this study was to identify suitable reference gene(s) for simultaneous normalization of gene expression in liver and adipose tissue of control and fructose-fed male rats which is considered as model of metabolic syndrome. Using TaqMan Real Time RT-PCR, we carried out an extensive evaluation of five frequently used endogenous controls, b-actin, b2-microglobulin, 18S rRNA, hypoxanthine phosphoribosyl transferase 1 and TATA box binding protein, for their presumed stability of expression. Expression stability was analyzed by GeNorm and NormFinder software packages, and by direct comparison of Ct values. Both, GeNorm and NormFinder identified 2-microglobulin in the liver, hypoxanthine phosphoribosyl transferase 1 in the adipose tissue and TATA box binding protein for comparative analysis of the two tissues as most stable genes. The combination of b-actin and TATA box binding protein in the liver, hypoxanthine phosphoribosyl transferase 1 and TATA box binding protein in the adipose tissue and b-actin and TATA box binding protein for both tissues together appeared to be the most suitable combinations of reference genes for studying alterations in gene expression in fructose-fed rats.