Exorneasy serum plasma kit

Frozen serum samples were incubated at 37ºC in a water bath until samples were completely thawed. The thawed samples were excluded cellular material, including thrombocyte fragments and particles larger than 0.8 µm using syringe filters (0.8 µm filter pore size, Millipore). Then removing some of the larger vesicles by passing through 0.22 µm filter (Millipore). Exosomes were isolated from pre‐filtered serum by exoRNeasy Serum/Plasma Kits (Qiagen).

Exosomal RNA was isolated by exoRNeasy Serum/Plasma Kits (Qiagen) according to the manufacturer's instructions.³⁰ Briefly, 800 μL pre‐filtered serum was mixed with equal volume buffer XBP. The sample/XBP mix was added onto the exoEasy spin column and spin the device for 1 minute at 500 × g. Buffer XWP was added onto the exoEasy column and spun 5 min at 5000 × g. 700 µl of QIAzol was added onto the membrane of exoEasy column, spun for 5 min at 5000 × g to collect the lysate, then 1 µl Caenorhabditis elegans miR‐39 (cel‐39, 1 μM) were added into the lysate for further external control. Subsequently, 90 µl of chloroform was used for phase separation. The upper aqueous phase/ethanol mix, buffer RWT and buffer RPE were added into RNeasy MinElute spin column in sequence as specified in the manufacturer's instructions. Finally, RNA was eluted in 20 µl RNase‐free water. RNA integrity and size distributions were evaluated by the Agilent 2100 Bioanalyzer Instrument.

The RNA content of PSC‐EVs and parent PSCs was detected by total RNA sequencing as previously described.²⁹ Briefly, total RNA was extracted from PSCs by Trizol (Life Technologies Corporation, Carlsbad, California). PSC‐EV‐derived RNA was purified using exoRNeasy Serum Plasma Kits (Qiagen, Hilden, Germany) following the manufacturer's guidelines. The RNA samples were quantified by deep sequencing with the Illumina NextSeq 500 platform (Illumina, San Diego, California). Data were analyzed using software packages including CLC Genomics Server and Workbench (RRID:SCR_017396 and RRID:SCR_011853), Partek Genomics Suite (RRID:SCR_011860), Spotfire DecisopnSite with Functional Genomics (RRID:SCR_008858).

EV RNA was extracted from 1.5 mL of plasma by using an exoRNeasy Serum/Plasma kit (Qiagen, Hilden, Germany) according to the manufacturer’s instruction. Exogenous spike-in cel-miR-39-3p was added to each sample during the extraction as loading control. After isolation, samples were cleaned and concentrated (Zymo Research, Irvine, CA, USA), then concentrations were measured with Qubit (Thermo Fisher Scientific, Waltham, MA, USA). All samples were loaded into a NanoString human miRNA panel (NanoString, Seattle, WA, USA) to measure the miRNA expression at pre-and post- MD intervention. miRNA expression level of each sample was normalized with the expression value of spike-in and housekeeping genes on nSolverTM (NanoString, Seattle, WA, USA). We presented the characteristics of plasma EVs in Appendix AFigure A1 and effects of MD on EVs characteristics in Appendix AFigure A2. The sizes and concentrations of EVs were not significant in both groups.

We followed the protocol in the Qiagen exoRNeasy Serum/Plasma kit (Cat No./ID: 77044) to capture extracellular vesicles on an affinity membrane, and then to lyse the vesicles and isolate the extracellular RNA. Briefly, 1 ml plasma samples were thawed at room temperature and centrifuged at 3,000 x g for 5 min at 4°C to remove debris (in an Eppendorf 5804 R centrifuge using an A-4-44). Samples were then applied to the column following the manufacturer instructions. This kit and the EVs isolated have been previously characterized (Enderle et al., 2015 (link); Tang et al., 2017 (link); Srinivasan et al., 2019 (link); Kalani et al., 2020 (link)).