RNA Extraction and qRT-PCR Protocol

Cells were lysed in RA1 buffer supplemented with 1% (v/v) β-mercaptoethanol (Macherey-Nagel) and stored at −80°C until processing. Total RNA was extracted by using the NucleoSpin RNA Extraction Kit (Macherey-Nagel) and the residual genomic DNA was removed by DNase digestion with RNase-free DNase (Macherey-Nagel). The total RNA quantifications were carried out using a NanoDrop spectrophotometer (NanoDrop Technologies). For cDNA synthesis, total RNA (100 ng) was reverse transcribed using the 5X iScript reverse transcription supermix (Biorad) according to the manufacturer's instructions. cDNA samples were stored at −20°C until use. Reverse transcription-quantitative PCR assays were performed in a LightCycler 480 instrument (Roche). Four microliter of 10-fold diluted cDNA were added to a mixture of (2X) iTaq Universal SYBR Green Supermix (Biorad) and 0.25 μM of each primer in a total volume of 10 μl. Thermal protocol was 95°C for 5 min followed by 40 cycles of 95°C for 10 s, 60°C for 30 s, and acquisition of a melting curve at the end of the run. The specificity of primer pairs was checked via melting curve analysis. Primers used in this study (Supplementary Table 1) were designed and tested as previously described (14 (link)). Fold changes were calculated by the ΔΔCt method using ACTB, PPIA, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as reference genes (15 (link)).