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.
Highlights
Real-time quantitative chain reaction relative or absolute analysis requires appropriated endogenous gene as reference gene for data normalization, which are known by: housekeeping gene, normalization gene, endogenous control gene, internal reference gene and suitable reference genes [1]
The results related to fractional quantitative Polymerase Chain Reaction (qPCR) cycles are represented as follow: 1) Hypothalamus (Figure 1)-Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) showed the lowest standard deviation (± 0.69), followed by Actb (± 1.27), Hypoxanthine phosphoribosyltransferase-1 (Hprt1) (± 1.35), Tubulin (± 1.69) and 18s (± 2.05); 2) Adrenal gland (Figure 2)-results showed that Hprt1 had the lowest standard deviation (± 0.96) followed by Gapdh (± 1.25), Actb (± 1.33), Tubulin (± 1.87) and 18s (± 1.92); 3) Fat tissue (Figure 3), Gapdh showed the lowest variation Cycle quantification (Cq) (± 1.52), followed by Hprt1 (± 1.65), Tubulin (± 2.26), 18s (± 2.48) and Actb (± 2.83)
In hypothalamus, when comparing all experiments groups, CT vs. D, CT vs. D or CT vs. times/wk during wk (TD) the most stable gene was 18s gene followed by Actb, Tubulin, Hprt1 and Gapdh genes
Summary
Real-time quantitative chain reaction (qPCR) relative or absolute analysis requires appropriated endogenous gene as reference gene for data normalization, which are known by: housekeeping gene, normalization gene, endogenous control gene, internal reference gene and suitable reference genes [1]. There are no studies concerning validation of reference genes to analyze the effects of resistance exercise (REx) and androgenic anabolic steroids (AAS) use on gene expression. Considering the increasing number of exercise-related and/or anabolic steroids articles, validation of the most stable reference genes for qPCR was considered of interest. 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
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