Hsa mir 16 5p

The purification and extraction of total cellular miRNAs were performed using the miRNeasy Mini Kit (Qiagen, Hilden, Germany). The extracted miRNAs were retro-transcribed by the miScript Reverse Transcription Kit (Qiagen, Germany), and the corresponding cDNA was diluted 1:3 in water. The miScript SYBR-Green PCR kit (Qiagen, Germany) was used to carry out qPCR in triplicate. Signals were detected on the MiniOpticon CFX 48 real-time PCR Detection System (Bio-Rad, Hercules, CA, USA). MiScript Primer Assays specific for hsa-miR-214-3p (MIMAT0000271), hsa-miR-16-5p (MIMAT0000069), hsa-miR-193a-3p (MIMAT0000459) and hsa-SNORD6 were purchased from Qiagen. The miRNA expression was measured using the 2^−ΔΔCt, and the SNORD6 gene was used as housekeeping.

We performed qPCR to evaluate the expression levels in a larger cohort from 26 Pca and 10 BPH EV RNA samples. Complementary DNA (cDNA) was prepared using Qiagen miRCURY LNA RT Kit (Hilden, Germany; cat. #339340) following the manufacturer’s protocol. This was followed by a real-time PCR expression analysis of the miRNAs identified at the initial experimental phase. The miRCURY LNA miRNA PCR Assay used are commercially available from Qiagen: (hsa-miR-16-5p, stock code: QIA/339306_YP00205702; hsa-miR-10a-5p, Stock code: QIA/339306_YP00204778; hsa-miR-194-5p, stock code: QIA/339306_YP00204080; hsa-miR-144-5p stock code: QIA/339306_YP00204670; hsa-miR-93-5p, stock code: QIA/339306_YP00204715; hsa-miR-326, stock code: QIA/339306_YP00204512; hsa-miR-221-3p, stock code: QIA/339306_YP00204532; hsa-miR-21-5p, stock code: QIA/339306_YP00204230). Real-time PCR was performed on the LightCycler480 system (Roche Diagnostics, Mannheim, Germany) using the absolute quantification method. The absolute copy number of cDNA was calculated using the standard curve prepared with qPCR experiments.