RNA Isolation and RT-qPCR Analysis of Gene Expression

Total RNA was isolated from cells and patients using TRIzol reagent (Thermo Fisher) according to the manufacturer’s instructions. A quantity of 2 μg of RNA was treated with the DNase Amplification Grade I Kit (Thermo Fisher) and reverse transcribed into cDNA using the Superscript-III kit (Thermo Fisher) following the manufacturer’s protocol. RT-qPCR was performed with SYBR Green Master Mix (Thermo Fisher) in a Rotor-Gene Q (Qiagen) under previously reported conditions [17 (link)]. The primers used are described in Supplementary Table S1. Mean values of ACTB and GAPDH housekeeping genes were used as the reference expression for the mRNA levels. Each sample was examined in triplicate. Fold expression was calculated according to the ΔΔC_t method [51 (link)]. ΔΔCt was calculated against expression in ShCTRL for experiments using cells, while ΔΔCt was calculated against the median of expression in the Luminal group for experiments using BC samples. For survival analysis, patients were classified into high and low-expression groups with the median fold-change for each gene considered to be the cutoff.