Mir 342 3p

Total microRNA was extracted and purified using the miRVana miRNA Isolation kit according to manufacturer’s instructions. RNA (5 μl) was converted into cDNA using the MicroRNA Reverse Transcription (RT) kit and specific RT primers (Thermofisher Scientific). Total RNA (500 ng or 1 μg) was reverse transcribed into cDNA using qScriptTM cDNA SuperMix (Quanta Biosciences, Beverly, MA). cDNA from cells was mixed with PerfeCTa SYBR Green FastMix ROX (Quanta Biosciences, Beverly, MA) or Taqman Universal Mix II No UNG plus specific PCR primers; AKAP12 (Qiagen, Germantown, MD), and TaqMan Assays [miR-106b-5p (Assay # 000442), miR-302b-3p, (Assay # 000531), miR-520e (Assay # 001119), miR-342-3p (Assay # 002260), miR-186-5p (Assay # 002285), miR-885-5p (Assay # 002296)] for qPCR using the Step Up Real PCR system (Applied Biosystems, Waltham, MA). Relative expression of mRNA and miRNA was normalized to GAPDH and snoRNA U44 expression and calculated using the 2-∆∆Ct method. Experiments were repeated three times and in triplicate.

miRNAs were determined using two-step RT-qPCR with RT-primer specific assay in combination with TaqMan probes: miR-143-5p (Assay ID: 463509_mat), miR-140-5p (Assay ID: 001187) miR-148a-5p (Assay ID: 473012_mat), miR-450a-5p (Assay ID: 462729_mat), miR-21-5p (Assay ID: 000397), miR-146b-5p (Assay ID: 002755), miR-342-3p (Assay ID: 002260), and miR-223-3p (Assay ID: 07896_mat) (Applied Biosystems; CA, USA). Each RT-reaction used 1.5 µL from the 14 µL eluted RNA using the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems; CA, USA). The RT reaction program and PCR cycling conditions were as we previously reported (Brianza-Padilla et al., 2016 (link)). miRNAs relative concentrations were normalized with Ct values of cel-miR-39, and values were calculated using 2^−ΔΔCt and 2^−ΔCt formulas. All Ct values for cel-miR-39 ranged from 20 to 22 cycles both for total plasma and for EVs RNA isolations.