Quantitative Real-Time PCR for Gene Expression

Total RNA was extracted from the cell samples using Trizol reagent (Invitrogen, Carlsbad, CA, USA) or in alternative Maxwell RSC simplyRNA Cells (Promega) and was processed with a Maxwell RSC Instrument (Promega) according to the manufacturer’s instructions. A reverse transcription reaction was performed using the Quantitect Reverse Transcription kit (Qiagen) following the manufacturer’s directions. A quantitative Real-Time PCR (qRT-PCR) was performed in LightCycler 96 (Roche, Penzberg, Germany) using LightCycler FastStart DNA Master SYBR Green I (Roche) and each validated primer. The validated qRT-PCR primers were from Eurofins (Milan). GAPDH was used as an internal control. Total RNA from formalin fixation and paraffin embedding (FFPE) tissues was isolated using the Maxwell RSC RNA FFPE kit (Promega, Madison, WI, USA) and was processed with a Maxwell RSC Instrument (Promega, Madison, WI, USA) as described in the study of Barbato et al. [65 (link)]. The primer sequences are listed in Table 2.