2.9.3. cDNA Synthesis and qPCR Parameters

First-strand cDNA templates for qPCR were synthesized in 20 μL reactions from 1 μg of DNaseI-treated, column-purified total RNA using random primers (250 ng; Invitrogen/Thermo Fisher Scientific), dNTPs (0.5 mM final concentration; Invitrogen/Thermo Fisher Scientific), M-MLV reverse transcriptase (200 U; Invitrogen/Thermo Fisher Scientific) with the manufacturer’s first strand buffer (1× final concentration) and DTT (10 mM final concentration) at 37 °C for 50 min.

PCR amplifications were performed in 13 μL reactions using 1× Power SYBR Green PCR Master Mix (Applied Biosystems/Thermo Fisher Scientific), 50 nM of both the forward and reverse primers, and the indicated cDNA quantity (see below). Amplifications were performed using the QuantStudio 6 Flex Real Time PCR system (384-well format) (Applied Biosystems/Thermo Fisher Scientific). The real-time analysis program consisted of 1 cycle of 50 °C for 2 min, 1 cycle of 95 °C for 10 min and 40 cycles of 95 °C for 15 s and 60 °C for 1 min, with fluorescence detection at the end of each 60 °C step and was followed by dissociation curve analysis.