Identification of Optimal Reference Genes for qRT-PCR Normalization for Physical Activity Intervention and Omega-3 Fatty Acids Supplementation in Humans.

The quantitative polymerase chain reaction (qRT-PCR) technique gives promising opportunities to detect and quantify RNA targets and is commonly used in many research fields. This study aimed to identify suitable reference genes for physical exercise and omega-3 fatty acids supplementation intervention. Forty healthy, physically active men were exposed to a 12-week eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) supplementation and standardized endurance training protocol. Blood samples were collected before and after the intervention and mRNA levels of six potential reference genes were tested in the leukocytes of 18 eligible participants using the qRT-PCR method: GAPDH (Glyceraldehyde-3-phosphate dehydrogenase), ACTB (Beta actin), TUBB (Tubulin Beta Class I), RPS18 (Ribosomal Protein S18), UBE2D2 (Ubiquitin-conjugating enzyme E2 D2), and HPRT1 (Hypoxanthine Phosphoribosyltransferase 1). The raw quantification cycle (Cq) values were then analyzed using RefFinder, an online tool that incorporates four different algorithms: NormFinder, geNorm, BestKeeper, and the comparative delta-Ct method. Delta-Ct, NormFinder, BestKeeper, and RefFinder comprehensive ranking have found GAPDH to be the most stably expressed gene. geNorm has identified TUBB and HPRT as the most stable genes. All algorithms have found ACTB to be the least stably expressed gene. A combination of the three most stably expressed genes, namely GAPDH, TUBB, and HPRT, is suggested for obtaining the most reliable results.

Effect of six weeks 1000 mg/day vitamin C supplementation and healthy training in elderly women on genes expression associated with the immune response - a randomized controlled trial.

BACKGROUND: In this study, we investigated the effects of supplementation and exercise on the expression of genes associated with inflammation like CCL2, CRP, IL1, IL6, IL10 mRNA in elderly women. METHODS: Twenty four participants divided randomly into two groups were subjected to 6 weeks of the same health training program (three times per week). SUP group (supplemented, n = 12, mean age 72.8 ± 5.26 years and mean body mass 68.1 ± 8.3 kg) received 1000 mg of Vitamin C/day during the training period, while CON group (control, n = 12, mean age 72.4 ± 5.5 years and body mass 67.7 ± 7.5 kg) received placebo. RESULTS: No significant changes in IL-1, IL-6, IL-10 and CRP mRNA were observed within and between groups. However, there was a clear tendency of a decrease in IL-6 (two-way ANOVA, significant between investigated time points) and an increase in IL-10 mRNA noted in the supplemented group. A significant decrease in CCL2 mRNA was observed only in the CON group (from 2^0.2 to 2^0.1, p = 0.01). CONCLUSIONS: It can be concluded, that 6 weeks of supplementation and exercise was too short to obtain significant changes in gene expression in leukocytes, but supplementation of 1000 mg vitamin C positively affected IL-6 and IL-10 expression - which are key changes in the adaptation to training. However, changes in body mass, IL1 and CCL2 were positive in CON group. It is possible that Vitamin C during 6 weeks of supplementation could have different effects on the expression of individual genes involved in the immune response. TRIAL REGISTRATION: Retrospectively registered.

Exercise Training and Vitamin C Supplementation Affects Ferritin mRNA in Leukocytes without Affecting Prooxidative/Antioxidative Balance in Elderly Women.

Physical training and antioxidant supplementation may influence iron metabolism through reduced oxidative stress and subsequent lowering of mRNA levels of genes that are easily induced by this stress, including those responsible for iron homeostasis. Fifteen elderly women participated in our 12-week experiment, involving six weeks of training without supplementation and six weeks of training supported by oral supplementation of 1000 mg of vitamin C daily. The participants were divided into two groups (n = 7 in group 1 and n = 8 in group 2). In group 1, we applied vitamin C supplementation in the first six weeks of training, while in group 2 during the remaining six weeks of training. In both phases, the health-related training occurred three times per week. Training accompanied by vitamin C supplementation did not affect prooxidative/antioxidative balance but significantly decreased ferritin heavy chain (FTH) and ferritin light chain (FTL) mRNA in leukocytes (for FTH mRNA from 2^64.24 to 2^11.06, p = 0.03 in group 1 and from 2^60.54 to 2^16.03, p = 0.01 in group 2, for FTL mRNA from 2^20.22 to 2^4.53, p = 0.01 in group 2). We concluded that vitamin C supplementation might have caused a decrease in gene expression of two important antioxidative genes (FTH, FTL) and had no effect on plasma prooxidative/antioxidative balance.