RT-PCR Assay for Transcript Expression

The expression levels of selected transcripts were confirmed by real-time polymerase chain reaction (RT-PCR) using absolute SYBR green ROX Mix (ThermoFisher Scientific, Rochester, NY, USA) as previously described [52 (link)]. OVCAR-8 and NCI/ADR-RES cells were treated with NCX4040 (5 µM) for 4, 24, 48 h in the complete media. Following treatment, cells were washed once with PBS (pH 7.4) and RNA was extracted with TRIzol (Ambion Life Technology, Grand Island, NY, USA). RNA was isolated and purified with RNA easy mini kit columns (Qiagen, Valencia, CA, USA). Data were analyzed using ΔΔCt method of relative quantification in which cycle times were normalized to β-actin (or GADPH) from the same sample. Primers for the selected genes were designed using Primer Express 1.0 software and synthesized by Integrated DNA Technologies, San Diego, CA, USA) and in some cases were purchased from Origene Technologies (Gaithersburg, MD, USA). All real-time fluorescence detection was carried out on an iCycler (Bio-Rad, Hercules, CA, USA).