Extracellular miRNA Profiling Protocol

Five milliliters of cleared secretomes were 1:1 diluted in PBS and ultracentrifugated (100,000× g, 9 h at 4 °C). After addition of exogenous Arabidopsis thaliana ath-miR-159a (30 pg) synthetic miRNA spike to evaluate RNA recovery and cDNA synthesis, total RNA was extracted with miRNeasy and RNeasy Cleanup Kits (Qiagen, Hilden, Germany), as previously reported [93 (link)]. The expression of 754 miRNAs was evaluated with the OpenArray system (Life Technologies, Foster City, CA, USA) with 384-well OpenArray plates, according to the manufacturer’s instructions. Each single miRNA was considered as present and considered for further analyses only when amplification appeared in all three samples. ath-miR-159 spike-in C_RT was used for the equalization of technical differences. Eventually, the global mean method [93 (link)] allowed the normalization between samples.