miRNA Expression Profiling by RT-qPCR

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.