Quantifying Gene Expression through qPCR

RNA from cell lines was extracted using illustra™ RNAspin Mini Isolation Kit (GE Healthcare, Bilbao, Spain) according to manufacturer’s instructions). Cells were washed twice with PBS and then 350 μL of RA1 lysis buffer with β-mercaptoethanol added. RNA purity and quantity were measured using a Nanodrop spectrophotometer and 2 μg of total RNA was transcribed using M-MLV Reverse Transcriptase and RNaseOUT (Life Technologies). Quantitative PCR was performed using PerfeCTa SYBR^® Green Supermix, Low Rox (Quanta, Barcelona, Spain) in a Viia7 Real-Time PCR System (Applied Biosystems, Madrid, Spain) with the following conditions: Taq polymerase activation 95 °C 3 min, denaturation 95 °C 15 s, annealing/extension 62 °C 1 min, melting curve 95 °C 15 s, 60 °C 1 min, 95°C 15 s, 40 cycles. The ∆∆Ct quantitation method was used to determine mRNA fold changes in gene expression, with 36B4 as the housekeeping gene. Sequences of primers are published [15 (link),48 (link),49 (link)].