Real-time quantitative PCR analysis of pre-treatment serum microRNAs from patients with prostate cancer

The overall goal of the study was to determine if serum or serum EV microRNAs added prognostic value to current clinical nomograms for prostate cancer. Serum was collected from 203 patients with biopsy-proven prostate cancer. EVs were isolated from the 132 serum samples. RNA was isolated from these serum and serum EV samples, and 61 microRNAs were quantified per sample on a custom qPCR plate. The microRNA data was normalized using NormFinder. The normalized microRNA data was then compared to adverse pathology and prostate biopsy Gleason grade group. Serum was collected in BD gold top (serum-separator) vacutainers from each patient and separated by centrifugation. Extracellular vesicles (EVs) were isolated from 500 µL using ExoQuick (System Biosciences). RNA was isolated from 200 µL serum per sample or the EV pellet using miRNeasy Serum/Plasma kit (Qiagen). RNA was quantified by Nanodrop and Qubit microRNA Assay. 5 µL of RNA was input for each cDNA reaction using the miRCURY LNA RT Kit (Qiagen). 61 microRNAs were quantified per sample on a custom PCR plate using the miRCURY LNA miR SYBR Green PCR Kit/Handbook (Qiagen). The samples were run on the Applied Biosystem ViiA7 instrument for 40 cycles.