EV Small RNA Profiling by OpenArray

Total RNA was isolated from similar EV numbers (10 ± 3 E9) using the miRNeasy Kit and RNeasy Cleanup Kit (Qiagen, Hilden, Germany), following the manufacturer’s instruction. To monitor the RNA recovery procedure, before RNA extraction, samples were spiked-in with exogenous ath-miR-159a (30 pg), an Arabidopsis thaliana synthetic miRNA whose specific primers are provided in the RT and PreAmp primer pools (Life Technologies, Foster City, CA, USA). miRNA cDNA samples were prepared by standard reverse transcription (RT) and preamplification procedures, as previously reported [64 (link)]. Preamplified samples were stored at −20 °C until expression analysis with the OpenArray system (Life Technologies) performed into 384-well OpenArray plates, according to manufacturer’s instructions. For each amplification curve, an AmpScore value was obtained. miRNAs with AmpScore < 1.1 or missing and Ct values > 27 were considered unamplified. The following assays (Life Technologies) were considered: hsa-miR-23a-3p 000399; hsa-miR-221-3p 000524; hsa-miR-423-5p 002340; hsa-miR-16-5p 000391; hsa-miR-26a-5p 000405; hsa-miR-103a-3p 000439; hsa-miR-101-3p 002253; hsa-let-7a-5p 000377; hsa-miR-425-5p 001516; U6 snRNA 001973; hsa-miR-146a-5p 000468; ath-miR159a 000338.