Perfecta multiplex qpcr toughmix

Digital PCR was performed in Sapphire chips on the Naica Crystal Digital™ PCR System (Stilla Technologies, Villejuif, France). The mastermix contained 1 × PerfeCTa Multiplex qPCR ToughMix (Quanta Bio, Beverly, USA) with 100 nM fluorescein, 800 nM of each primer and 250 nM hydrolysis probe for each target (TTC17 and EqPH-V) and 9 µL template. Following droplet generation, amplification was carried out according to the following protocol: 95 °C for 10 min, 45 cycles of 95 °C for 10 s and 60 °C for 40 s. Endpoint fluorescence values measured on the Naica Prism 3 reader were analysed by Crystal Miner software (v. 2.4.0.3; Stilla Technologies, Villejuif, France). The software’s standard settings were applied to export the copy numbers for the two targets.

To preliminarily verify the diagnostic value of urine EV DNA, we selected RASSF1A, a gene widely recognized for its hypermethylation in PCa (Connell et al., 2019 (link)), and performed methylation analysis by Droplet digital PCR (ddPCR). This detection was performed using the Naica^® six-channel microdrop chip digital PCR system from Stilla technologies (France). PCR reactions were prepared with 10 ng sulfite-conversed DNA using the PerfeCTa Multiplex qPCR ToughMix (Quanta bio) within Sapphire chips (primers, probes, reaction system and amplification conditions listed in Supplementary Material S2). ddPCR was performed using a Naica Geode, programmed to partition the sample into droplets, followed by the thermal cycling procedure as suggested in the user’s manual. Images were acquired using a Naica Prism3 Reader and analyzed using Crystal Reader software for total droplet enumeration and droplet quality control, along with Crystal Miner software for extracted fluorescence values for each droplet.

Total RNAs were isolated from brain regions and peripheral tissues using the TRIzol Reagent. Single strand cDNAs were synthesized using qScript XLT cDNA SuperMix (Quantabio, Beverly, MA, USA, #95161-500). Rodent isoform CB1A [3 (link)] and CB2A [4 (link)] FAM-labeled probes and endogenous control VIC-labeled Actb probe (Thermo Fisher Scientific Inc., Waltham, MA, USA, #4352341E) were used for TaqMan RT-qPCR. To validate hepatocyte CB1R detection, TaqMan PreAmp Master Mix Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA, #4391128) or PerfeCTa PreAmp SuperMix (Quantabio, Beverly, MA, USA, #95146-005) was used for cDNA preamplification using 80 nM of forward and reverse primer sets [48 (link)]. cDNAs were pre-amplified using the program: 95 °C hold for 10 min and then 10 cycles of denaturation at 90 °C for 15 s and annealing and extension at 60 °C for 4 min. Duplex PCR assays containing both the target and endogenous control TaqMan probes were carried out with Advanced TaqMan Fast PCR Master Mix (Thermo Fisher Scientific Inc., Waltham, MA, USA, #4444556) or PerfeCTa Multiplex qPCR ToughMix (Quantabio, Beverly, MA, USA, #95147-250) in StepOnePlus instrument using a default thermo-cycling program. The relative fold change is calculated using the formula: 2^{(−△△Ct)} [48 (link)].