RNA Extraction and miRNA Expression Analysis

miRNeasy and RNeasy Cleanup Kits (Qiagen, Hilden, Germany) were used to extract total RNA from similar iASC-EV numbers (26 ± 4 E9, mean ± SEM), following the manufacturer’s instruction. To monitor the process of RNA recovery and cDNA synthesis, before RNA extraction samples were spiked-in with exogenous ath-miR-159a (30 pg), an Arabidopsis thaliana synthetic miRNA. Specific primers for ath-miR-159a were provided in the RT and PreAmp primer pools (Life Technologies, Foster City, CA, USA). cDNA samples were prepared by standard reverse transcription (RT) and pre-amplification procedures, as previously reported [34 (link)]. Expression analysis with the OpenArray system (Life Technologies) was performed into 384-well OpenArray plates, according to the manufacturer’s instructions. An AmpScore value was obtained for each amplification and candidates with AmpScore <1.1 or missing, together with Crt values >25 were considered unamplified. When unamplified, an arbitrary Crt value of 25 was assigned. All assays for RGs and OA-miRNAs were purchased from Life Technologies.