2.10 Relative gene expression by quantitative polymerase chain reaction (qPCR)
Total RNA from U-87 MG cells, male rat neonatal cardiomyocytes, male murine adult ventricular cardiomyocytes and male rat thoracic aorta was extracted with TRIzol reagent (Thermo-Fisher, Waltham, MA, USA) and cDNA was synthesized using First Strand cDNA Synthesis (Thermo-Fisher, Waltham, MA, USA) kit following the manufacturer’s instructions. qPCR was performed in a StepOnePlus Real Time PCR system (Applied Biosystem, Beverly, MA, USA) using Maxima SYBR PCR 2x (Thermo-Fisher, Waltham, MA) and primers (Table 1) (Síntese Biotecnológica, Belo Horizonte, Brazil) at the final concentration of 600 nM. The cDNA was diluted in RNAse free water 1:100. Thermal cycling protocol was as follows: i) denaturation at 95°C for 5 minutes followed by; ii) 45 cycles of denaturation at 95°C for 10 seconds; iii) annealing/extension at 60°C for 1 minute. Relative expression of the selected genes was performed by 2-ΔΔCt method (Livak et al. , 2001) using the housekeeper gene S26 as normalizer.