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Andreo Fernando AguiarQuantitative analyses of gene expression by RT-qPCRRNA extraction. Total RNA was extracted from muscle samples with TRIzolReagent (Life Technologies, Carlsbad, CA, USA), which is based on the guanidinethiocyanate method. Frozen muscles were mechanically homogenized in 1 mL ofTRIzol reagent. Total RNA was resuspended in RNase-free water, treated with DNase I(Life Technologies, Carlsbad, CA, USA) to remove any possible DNA present insample. Total RNA was quantified using Nanodrop spectrophotometer (AgilentTechnologies, Santa Clara, CA, USA), measuring optical density (OD) at 260nm. RNApurity was ensured when OD260/280 ratio was around 2.0. And RNA integrity wasverified by 1% agarose gel electrophoresis, which well defined bands were observedcorrespondents to 18S and 28S ribosomal RNAs.cDNA synthesis. cDNA was synthesized from 2µg of total RNA using HighCapacity cDNA archive kit (Life Technologies, Carlsbad, CA, USA). Each reaction had10µL of 10X Reverse Transcription Buffer, 4µL of 25X dNTPs, 10µL of 10X randomprimers, 100 units of RNase inhibitor (Life Technologies, Carlsbad, CA, USA), 250units of Reverse Transcriptase MultiScribe, and the final volume was adjusted to100µL with nuclease-free water. And cDNA synthesis reaction conditions were: 10minutes at 25°C for primers annealing and 2 hours at 37°C for reverse transcription.Reaction control was made by omission of the reverse transcriptase enzyme. All cDNAsamples were amplified by PCR to ensure that there was no contamination byDNA. The resulting cDNA samples were aliquoted and stored at -20°C. 2µL of cDNA,corresponding to 20ng of total RNA, were used as template in real time PCR reactionswhich were made in 7300 Real-Time PCR System (Life Technologies, Carlsbad, CA,USA) with equipment universal thermal cycling conditions: 95°C for 10min, 40 cyclesof 95°C for 15s and 60°C for 1min. The reactions were performed in duplicate using 0.4µM of each primer and 2X Power SYBR Green PCR master mix (Life Technologies,Carlsbad, CA, USA) with final volume of 25 µL.Quantitative real-time RT-PCR. Series of five dilutions (10x) was constitutedfrom PCR products diluted 500 times, for each gene, from an initial mixture containingequal amounts of cDNA from three extra samples used only for standardization, thuswere generated a five-point standard curve for each primer set initially chosen forqPCR. qPCR linearity and efficiency were calculated from those standard curves slope,50