Which housekeeping gene is the best choice for RT-qPCR analysis in mice fed with a high-fat diet? Studies in the liver, kidney, pancreas, and intestines

Abstract

High-fat diet (HFD)-induced obesity in rodents in association with Reverse Transcription and real-time Polymerase Chain Reaction (RT-qPCR) analysis have been extensively used to investigate the gene expression regulation in metabolic syndrome. qPCR requires careful standardization, and the commonly chosen housekeeping genes (HKGs) are involved in basal metabolic pathways, which makes the choice of HKG more difficult in obesity-related studies. In this study, we investigated the expression and stability of putative HKGs, such as Ppia, Tubb2a, Tfrc, Tbp, Mrpl32, Gusb, Rn18s, Rplp0, Hprt-1, B2m, Gapdh and Actb in the liver, kidney, endocrine and exocrine pancreas, small and large intestines of HFD-treated mice. The Ct values were used to determine the stability of HKG expression by RefFinder, comparing the GeNorm, NormFinder, BestKeeper, and comparative delta-Ct method algorithms. The influence of the diet on putative HKGs expression was also evaluated by 2-ΔΔCt analysis. The most stable HKGs were: Tbp and Mrpl32 in the liver, Mrpl32 and Tubb2a in the kidney, Hrpt-1 and Gapdh in the pancreatic islets and cecum, Tbp and Ppia for the exocrine pancreas, and Rn18s, Hrpt-1, and Actb for jejunum. On the contrary, Tubb2a|Trfc must be avoided to normalize the qPCR data from the HFD liver, B2m from the kidney, Trfc|Ppia and Tubb2a|B2m from the endocrine and exocrine pancreas, respectively, B2m from the jejunum, and Rn18s and Actb from the cecum portion of the large intestine. We provided the best and worse HKGs to use in the RT-qPCR analysis of tissues involved in the regulation of energy metabolism in the HFD mice model.