Maxwell rsc simplyrna cells

Total RNA was extracted from treated cells with Maxwell®RSC simplyRNA Cells (Promega, Madison, Wisconsin, USA) and retrotranscribed with iScript cDNA kit (Bio-Rad, Hercules, California, USA). Quantitative Real-Time PCR (qRT-PCR) was performed using GoTaq® qPCR Master Mix (Promega, Madison, Wisconsin, USA) in a CFX96 Real Time PCR Detection System (Bio-Rad, Hercules, California, USA). Relative expression of target genes was calculated using the ΔΔCt method by normalizing to the reference gene expression Beta-DGlucuronidase (GUSB). For RNA-Seq validation normalization was performed with the geometric mean of three reference genes expression: Hypoxanthine Phosphoribosyltransferase 1 (HPRT), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), Ribosomal Protein S17 (RPS17). See Additional file 2: Table S2 for qRT-PCR primers sequences.

Total RNA was extracted with Maxwell®RSC simplyRNA Cells (Promega, Madison, WI, USA) and retrotranscribed with iScript cDNA kit (Bio-Rad, Hercules, California, USA). Quantitative Real-Time PCR (qRT-PCR) was performed using Sso Fast EvaGreen Super Mix (Bio-Rad, Hercules, California, USA) in a CFX96 Real Time PCR Detection System (Bio-Rad, Hercules, CA, USA). Relative expression of target genes was calculated using the ΔΔCt method by normalizing to the reference gene expression Cyclophillin A (CYPA). See Additional file 2 Table S1 for qRT-PCR primers.

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.