RNA was isolated from plasma, circulating MVs, exosomes, and vesicle‐free supernatant (ie, the remaining supernatant after exosome isolation) by using a TRIzol‐based miRNA isolation protocol. For each patient, 250 μL total plasma was diluted in 750 μL TRIzol LS (Life Technologies) to measure plasma miRNA levels. An additional 250 μL total plasma was centrifuged at 20 000g for 30 minutes at 4°C to pellet circulating MVs.18 (link) The pellet was diluted in 250 μL RNase‐free water and then diluted in 750 μL TRIzol LS to measure MV miRNA levels. Caenorhabditis elegans miR‐39 (cel‐miR‐39; 5 nmol/L; Qiagen) was spiked in TRIzol for normalization of miRNA content, as described previously.8 (link) To increase the yield of small RNAs, the RNA was precipitated in ethanol at −20°C overnight with glycogen (Invitrogen).
To analyze miRNA levels in exosomes and vesicle‐free supernatant, the remaining supernatant after 20 000g centrifugation was collected and centrifuged at 100 000g for 1 hour at 4°C to pellet exosomes.19 (link) The pellet was diluted in 250 μL RNase‐free water and then diluted in 750 μL TRIzol LS to measure exosome miRNA levels. The remaining supernatant after exosome isolation, defined as “vesicle‐free supernatant,” was diluted in 750 μL TRIzol LS and processed, as described.
To analyze miRNA levels in exosomes and vesicle‐free supernatant, the remaining supernatant after 20 000g centrifugation was collected and centrifuged at 100 000g for 1 hour at 4°C to pellet exosomes.19 (link) The pellet was diluted in 250 μL RNase‐free water and then diluted in 750 μL TRIzol LS to measure exosome miRNA levels. The remaining supernatant after exosome isolation, defined as “vesicle‐free supernatant,” was diluted in 750 μL TRIzol LS and processed, as described.
