RNA preparation and real time PCR
Total RNA was purified using Direct-zol RNA Miniprep (Zymo Research, Irvine, California, USA), according to the manufacturer’s instructions, including a step with deoxyribonuclease incubation. For real time PCR, RNA (150 ng) was reverse transcribed to cDNA with 8 U/µg RNA of Moloney murine leukemia virus reverse transcriptase (Promega, Milan, Italy) and random hexamer primers in a final volume of 25 μl; the reaction was performed at 37°C for 1 h, and the enzyme inactivated at 75°C for 5 min. Control reactions without the addition of the reverse transcription enzyme were performed (data not shown). Triplicates of 1:4 cDNA dilutions were amplified using GoTaq®qPCR Master Mix technology (Promega) according to the manufacturer’s protocol, with QuantStudio®3 real time PCR system (Applied Biosystems, Waltham, Massachusetts, USA) with the following thermal profile: 2 min at 95°C; 40 cycles, 15 sec at 95°C, 1 min at 60°C. Primer sequences are reported in Supplementary Table 1. Data were analyzed using the 2-ΔΔCt method.