Extracellular RNA Quantification Protocol

Five ml of cleared secretome was 1:1 diluted in PBS before ultracentrifugation at 100,000× g for 9 h at 4 °C. Resulting pellets were processed with miRNeasy and RNeasy Cleanup Kits (Qiagen, Hilden, Germany) after addition of 30 pg of exogenous Arabidopsis thaliana ath-miR-159a synthetic miRNA spike. This was used to evaluate RNA recovery and cDNA synthesis performed as previously reported [14 (link)]. The OpenArray system (Life Technologies, Foster City, CA, USA) was used to determine miRNA expression in 384-well OpenArray plates according to the manufacturer’s protocol. Each single miRNA was considered as present when amplification resulted in all three samples. Eventually, ath-miR-159 spike-in C_RT was used to equalize technical differences, and the global mean method [15 ] allowed normalization between samples.