2.5. cDNA synthesis, RT-PCR and RT-qPCR

The reverse transcription reactions were performed in a total volume of 20 μl using 0.5–2 μg of total RNA, 500 ng/μl Anchored Oligo-dT primers (Thermo Fisher Scientific), and 200 U Superscript III™ reverse transcriptase (Invitrogen) following manufacturer’s first-strand cDNA synthesis protocol. All cDNA samples were diluted in 9 volumes of sterile de-ionised water and stored at −20 °C until RT-qPCR analyses. Semi-quantitative RT-PCR reactions were carried out using PlatinumTaq (Invitrogen) in a C1000™ Thermal Cycler (Bio-Rad Laboratories, Hercules, CA, USA) for 35 cycles (30 s. denaturation at 94 °C followed by 30 s. annealing at 60 °C and 1 min extension at 72 °C). Quantitative PCR was performed with an iCycler iQ™5 Real-Time Detection System (Bio-Rad Laboratories) using 8 μl diluted cDNA, 0.4 μM of the forward (1 μl) and reverse (1 μl) primer, and 10 μl 2× SYBR-Green I dye master mix (QuantiTect^® SYBR^® Green PCR kit, Qiagen) in a reaction volume of 20 μl. The RT-qPCR reaction mix was denatured at 95 °C for 10 min and then subjected to 45 amplification cycles (10 s. denaturation at 95 °C, 45 s. annealing at 60 °C and 30 s. extension at 72 °C) [3]. Under these optimized qPCR conditions, each primer set presented amplification efficiency close to 100% (Fig. S4) and a single peak in melt-curve analyses.