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.