Exosome-Derived miRNA Isolation Protocol

Prior to miRNA extraction, exosomes were isolated from cell-conditioned media using the miRCURY Exosome Cell/Urine/CSF Kit (Qiagen, Hilden, Germany), and miRNA isolation was performed using the miRCURY RNA Isolation Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. To allow for normalization of sample-to-sample variation in the RNA isolation, prior to purification, each sample was spiked with UniSp2, UniSp4, UniSp5, each at a different concentration in 100-fold increments, with UniSp6 and cel-miR-39-3p as positive controls for cDNA synthesis. RNA quality was determined with the Agilent RNA 6000 Nano Kits using 2100 Bioanalyzer (Agilent Technologies, St. Clara, CA, USA). The degradation rate of total RNA was determined using RIN values. Only the samples with RIN > 7 were included in further analysis. Directly after isolation, RNA was subjected to reverse transcription [45 (link)].