cDNA synthesis and quantitative PCR (qPCR)

Total RNA (10 μg) isolated from cells was treated with 4 U TURBO™ DNase (Ambion) in the presence of RiboLock RNase Inhibitor (Thermo Fisher Scientific), according to the manufacturer's protocol. Phenol:chloroform extraction was then performed and RNA was precipitated with isopropanol. DNase-treated RNA (2 μg) was used for cDNA synthesis. Reverse transcription was carried out using a mixture of 50 pmol of an oligo(dT) primer, 250 ng of random primers (Invitrogen) and SuperScript III Reverse Transcriptase (Invitrogen) according to the manufacturer's guidelines, in a final volume of 20 μl. Samples were diluted 10-fold and mixed with 2.5 pmol of each qPCR primer (see Supplementary Table S2 for sequences), 0.5 μg bovine serum albumin and Platinum^® Quantitative PCR SuperMix-UDG (Invitrogen), in a final volume of 10 μl. qPCR was carried out in a Roche LightCycler^® 480 system. Negative controls (-RT) were included for each experiment and showed an insignificant background. GAPDH mRNA was used for normalization. Reaction specificity was confirmed by melting curve analysis. Analyses were conducted in triplicate.