Exorneasy mini kit

Total exosomal RNA was extracted from EVs using a ExoRNeasy mini-kit (Qiagen, Milan, Italy) according to the manufacturer’s instructions. Identification and quantification of miRNAs were undertaken with Small RNA-Seq (Sequentia Biotech SL, Barcelona, Spain). In brief, Illumina NGS libraries were prepared and sequenced using an Illumina HiSeq2000 sequence analyzer (Illumina Inc., San Diego, CA, USA). Before further analysis, a quality check was performed on the raw sequencing data, deleting low quality portions while preserving the longest high-quality part of NGS reads. The high-quality reads were aligned against the Homo sapiens genome (Ensembl GRCh38) with a STAR aligner (version 2.7.7a). STAR can align spliced sequences of any length with moderate error rates, providing scalability for emerging sequencing technologies. STAR generates output files that can be used for many downstream analyses, such as transcript/gene expression quantification. Out of the 734 miRNAs present in the official annotation, a differential expression analysis between the groups (MPV vs. CTL) was performed. 89 miRNAs were identified, 25 of them were dysregulated (2 upregulated and 23 downregulated).

Exosomes were isolated from plasma samples using ExoQuick Plasma prep and Exosome isolation kit (System Biosciences, CA, USA). We modified the manufacturer’s protocol for sample-optimized exosome isolation. The modified protocol was intended to avoid unnecessary sample waste and remove residual impurities. An ExoQuick reagent was used to precipitate plasma exosomes, while the ExoRNeasy mini kit (Qiagen) was used to directly purify RNA from exosomes. To isolate exosomes, 250 µL of the filtered plasma was added to 63 µL of ExoQuick (System Biosciences, CA, USA) and incubated for 30 min at 4 °C. The ExoQuick/plasma mixture was centrifuged at 1500 g for 30 min, followed by supernatant removal and centrifugation of the EV pellet at 1500 g for 5 min to remove the residual ExoQuick solution. The EV pellet was resuspended in 200 µL of phosphate-buffered saline, and the precipitated exosomes were used immediately.