Validation of Reference Genes for qPCR Analysis of Resistance Training and Androgenic Anabolic Steroids on Hypothalamus, Adrenal Gland and Fat Tissue

Abstract

Background: Real-time quantitative Polymerase Chain Reaction (qPCR) is a technique used for quantification of gene expression and the use of reference genes is very important to normalize the quantification results. Aim: To validate the most suitable reference genes for resistance exercise training (REx) and use of nandrolone decanoate (DECA) in three different rat tissues. Methods: A total of 40 adult male Wistar rats were distributed into four groups: exposed to vehicle three times per week (wk) (CT); eight wk of REx exposed to vehicle three times per wk (T); exposed to DECA three times per wk (D); eight wk of REx exposed to DECA three times per wk (TD). Stability of the following genes was evaluated: beta actin (Actb), alpha Tubulin (Tubulin), Glyceraldehyde-3-phosphate dehydrogenase (Gapdh), Hypoxanthine phosphoribosyltransferase-1 (Hprt1) and 18s Ribossomal RNA (18s) in hypothalamus, adrenal gland and mesenteric fat tissue using GeNorm, NormFinder and BestKeeper software. Results: In hypothalamus and adrenal, all genes were suitable and none was rejected by statistical analysis; however, in fat tissue, Actb, Gapdh and Hprt1 genes were rejected by geNorm but not the others two software. Conclusion: In hypothalamus and adrenal all selected genes analized were stable and can be used for qPCR gene expression analysis. However, in fat tissue we suggest the Tubulin gene as most stable gene.