Quantitative Analysis of AR Isoforms

For each RNA sample, 12 µL of the template were retrotranscribed to cDNA using a QuantiTect Reverse Transcription Kit (Qiagen, Venlo, The Netherlands) for a final volume of 20 µL. The assessment of AR-V7, AR-FL and RNY4 copies was performed on cDNA using custom assays (primers described elsewhere) [12 (link),14 (link)]. DNA was employed to detect the AR T878A variant (TaqMan™ SNP Genotyping Assay C_175239649_10, Thermo Fisher Scientific, MA, USA), AR gene copy numbers with RNaseP as the internal reference standard (TaqMan™ Copy Number Assay Hs04107225_cn and TaqMan™ Copy Number Reference Assay, human, RNase P, Thermo Fisher Scientific, USA). A volume of 15 μL of the reaction mix was loaded on a QuantStudio 3D Digital PCR 20K Chip (Thermo Fisher Scientific, MA, USA) and thermocycling ran at 95 °C for 8 min, 40 cycles (37 cycles for the AR-V7 assay) at 95 °C for 15 s and at 60 °C for 1 min, with a final extension step at 60 °C for 2 min. Copies/µL reaction were retrieved with QuantStudio 3D AnalysisSuite Cloud Software and copies/mL plasma were calculated by multiplying this value by the dilution factors applied during the process.