Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR)

Total RNA was extracted from T-47D cells using “Trizol” reagent (Thermo Fisher Scientific, Waltham, MA, USA) as per the manufacturer’s protocol. gDNA-free total RNA was reverse transcribed using iScript cDNA Synthesis Kit (Bio-Rad Laboratories Inc) according to the manufacturer’s instructions. Prior to RT-qPCR analysis, cDNA was diluted 10-fold in PCR-grade water. qPCR of reverse transcribed cDNA (RT-qPCR) was performed in 96-well format in the Bio-Rad CFX384 Real Time System. The assay included no template, no template during RT, and no RT controls to detect reagent contamination and presence of genomic DNA. The 96-well plates were arranged in randomized treatment maximization blocks. For data analysis, the quantification cycle (Cq) value was determined and specific gene expression normalized to endogenous control using ΔΔCq method. Expression of ACTB and HPRT-1 genes was set as endogenous controls (reference genes). The normalized ΔCq from treated samples was compared with the stripped control (Cs) to obtain ΔΔCq values and used to calculate relative fold change compared with control. ESR1 mRNA levels were determined by RT-qPCR. T-47D cells were treated in the presence or absence of 100 µM BC, E₂, and ICI 182, 780 (ICI) for 24 hours. Results are shown as the mean ± standard error of the mean (SEM) of at least three independent experiments with three replicates in each experiment.