Quantitative Analysis of Gene Expression

Six independent experiments with 3–4 HGC samples collected after 48-h treatment in the presence or absence of BPS (10 nM or 1 µM) were used for transcriptomic analysis using real-time quantitative polymerase chain reaction (qPCR). Briefly, total RNA was extracted from HCG using the TRI reagent and treated using XS RNA Nucleospin (Macherey Nagel, France), following the manufacturer’s instructions. Subsequently, the RNA concentration was determined using a NanoDrop ND-1000 spectrophotometer (Nyxor Biotech, Paris, France), and RNA integrity was checked using electrophoresis. DNAse treatment and reverse transcription (RT) was performed on 150 ng total RNA extracted from CC using the Maxima First Strand cDNA Synthesis Kit (ThermoFisher Scientific), according to the manufacturer’s recommendations.
qPCR reactions were performed as previously described [59 (link)]. The geometric mean of two housekeeping genes (ribosomal protein L19 [RPL19] and glyceraldehyde 3-phosphate dehydrogenase [GAPDH]) was used to normalise gene expression. The relative amounts of gene transcripts (R) were calculated according to the equation:
$\mathrm{R} = \frac{\left(E_{gene}^{-Ct gene}\right)}{\left(\mathrm{geometric} \mathrm{mean} \left(E_{GAPDH}^{-Ct GAPDH}; E_{RPL19}^{-Ct RPL19}\right)\right)}$ where E is the primer efficiency (Table 1) and Ct the cycle threshold.