Plasma AR Gain and Exosomal RNA Analysis

Peripheral blood samples were collected from each patient within 30 days before starting abiraterone or enzalutamide treatment. Overall, 10 ml of blood were collected, transferred in ethylene-diamine-tetra-acetic acid (EDTA) tubes and centrifuged at 1900 × g for 10 min at 4 °C within 2 h after drawing. Plasma was divided into 2 aliquots of 2 ml and stored at −80 °C until analysis. Circulating free DNA was extracted with the QIAamp Circulating Nucleic Acid Kit (Qiagen, Valencia, CA) and the total DNA was quantified using the Quant-iT high sensitivity PicoGreen double-stranded DNA Assay Kit (Invitrogen) or by spectrophotometric evaluation (NanoDrop® ND-1000, Milan, Italy). A multiplex digital droplet PCR (ddPCR; Bio-Rad, Hercules, CA) assay was performed to assess plasma AR gain, as previously described [9 (link)]. Four reference genes have been used: NSUN3, ElF2C1, AP3B1, and ZXDB at Xp11.21 as a control gene not involving the whole arm of chromosome and each PCR reaction was made with 1–2 ng DNA. AR gain was defined as copy number amplification when over the 2.01 threshold [9 (link)]. Exosomes-derived RNA extraction was performed using the exoRNeasy kit (Qiagen, Valencia, CA), RNA was transcribed into complementary DNA, amplified using the One-Step RT-ddPCR Kit (Bio-Rad, Hercules, CA) and analyzed by a digital droplet PCR, as previously described [15 (link)].