Exorneasy kit

Two samples of 9 mL EDTA blood were collected and stored for a maximum of 4 h at 4 °C. CTCs were isolated in duplicate from 5 mL of whole blood by positive immunomagnetic selection targeting EpCAM, EGFR, and HER2 (AdnaTest EMT-2/StemCell Select^TM, QIAGEN) [22 (link)]. The CTC-depleted blood remaining after positive immunomagnetic selection [23 (link)], as well as the remaining blood (not used for CTC isolation) were centrifuged at 1841× g for 8 min and the obtained plasma was frozen at −80 °C. EVs were isolated from 4 mL prefiltered (0.8 µm pore size) plasma by affinity-based binding to a spin column [22 (link),24 (link)]. Subsequently, the total RNA was isolated and purified (exoRNeasy Kit, QIAGEN). The mRNA was isolated from the CTC lysates and from the vesicular RNA eluates by Oligo(dT)₂₅ beads and was reverse transcribed (AdnaTest EMT-2/StemCell Detect^TM, QIAGEN) [22 (link)]. cfDNA was isolated by affinity-based binding to magnetic beads (QIAamp MinElute ccfDNA Kit, QIAGEN), as previously described [25 (link)], using plasma from CTC-depleted blood (≥1 mL, preferentially the maximal available volume; mean: 4.6 mL). cfDNA quantification was performed using the Agilent Chip High Sensitivity DNA assessing the concentration of all fragments with lengths between 100 and 700 bp.

Eighteen milliliters of EDTA blood was collected and CTCs were isolated in duplicate from 5 ml of whole blood by positive immunomagnetic selection (AdnaTest EMT-2/StemCell Select^TM, QIAGEN) [13 (link)]. CTC-depleted blood remaining after positive immunomagnetic selection [20 (link)] as well as the remaining blood (not used for CTC isolation) were centrifuged and stored. EVs were isolated from pre-filtered plasma by affinity-based binding to a spin column [13 (link), 21 (link)]. Subsequently, the total RNA was isolated and purified (exoRNeasy Kit, QIAGEN). The mRNA was isolated from the CTC lysates and from the vesicular RNA eluates by Oligo(dT)₂₅ beads [13 (link)]. The supernatant remaining from the CTC lysates after incubation with the Oligo(dT)₂₅ beads, called the mRNA-depleted CTC lysate, was used to isolate the gDNA using the AllPrep DNA/RNA Nano Kit prototype (QIAGEN) [14 (link)]. cfDNA was isolated by affinity-based binding to magnetic beads (QIAamp MinElute ccfDNA Kit, QIAGEN) using plasma from CTC-depleted blood [20 (link)]. Buffy coat DNA and normal tissue DNA that was available (from 18 of the 26 patients) was used as matched germline control. Detailed protocols are available in Additional file 2.

The ExoRNeasy kit (Qiagen) was used to isolate cell-free RNA from blood plasma of de-identified healthy controls (blood collected in K2EDTA tubes; Discovery Life Sciences) and patients with pancreatic cancer (blood collected in K2EDTA tubes; BioIVT). Samples were initially filtered through a 0.8 µm filter to remove any contaminants, such as buffy coat. Filtered plasma was then processed using the ExoRNeasy kit to isolate cell-free RNA according to manufacturer instructions.

EVs and EV-contained total RNA were isolated from biobanked urine and serum supernatants using the exoRNeasy kit (Qiagen, Düsseldorf, Germany) according to manufacturers’ instructions. Total RNA from serum supernatant was isolated with the miRNeasy kit (Qiagen). For the purpose of EV quantification, EVs were isolated from separate aliquots of urine and serum samples corresponding to the first sequencing set using the miRCURY Exosome Cell/Urine/CSF and miRCURY Exosome Serum/Plasma kits (Qiagen). Five milliliters of urine and 0.5 mL of serum supernatant were used for isolation for both kits. Prior to isolation, samples were centrifuged for 5 min at 3000× g in order to remove cryoprecipitates.

RNA from prefiltered plasma was isolated with exoRNeasy kit (Qiagen Hilden, Germany) according to the manufacturer's protocol. The miR-21 levels were tested by miScript PCR System (QIAGEN, Hilden, Germany) following extraction of RNA from 500 μl of plasma collected from mice treated with Curcumin and control mice, treated with vehicle (corn oil).

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)].