2.3. Sirtuin 1 Assessment
SIRT1 serum concentration was determined using an ELISA kit (Uscn Life Science, Wuhan, Hubei, China). Serum samples, before and after interventions, were analyzed in duplicate and in the same ELISA plate using the Multiscan FC plate reader (Thermo Scientific, Waltham, MA, USA), with a coefficient of variation of 12%, according to the manufacturer’s instructions. All tests were performed according to the manufacturer’s instructions.Gene expression of SIRT1 was evaluated at pre- and post-inclusion in the protocol by using the specific assay Hs01009005_m1 (Applied Biosystems, Life Technologies, 151, Waltham, MA, USA). Total RNA was obtained from peripheral leukocytes using the TRIzol reagent (Life Technologies). cDNA synthesis was performed using a Superscript II kit (Life Technologies) with 1 μg from total RNA in a final volume of 20 μL, according to the manufacturer’s instructions. Expression of the housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), used as the normalizing gene, was evaluated by using the specific assay # Hs02758991_g1 (Applied Biosystems). The reaction mix was prepared using 5 μL of Universal Master Mix (Life Technologies), 0.5 μL of primers and probes mix (×20), and 2.5 μL of cDNA diluted samples (1:5).PCR was performed according to the following protocol: enzymatic activation for 2 min at 50 °C, initial denaturation for 10 min at 95 °C, followed by 40 cycles of denaturation for 15 s at 95 °C and annealing for 20 s at 60 °C. Reactions were run in triplicate, and the number of copies of the gene transcript was determined using Ct (“threshold cycle”) values. For calculations, Ct values of SIRT1 were subtracted from Ct values for the GAPDH gene. The results are expressed in arbitrary units (AU).