Mirneasy micro kit

RNA from exosomes was extracted using the SeraMir Exosome RNA Purification Column Kit (System Biosciences) following the manufacturer's protocol. RNA from BM cells was extracted using the miRNeasy Micro Kit (QIAGEN) following the manufacturer's protocol. miRNA was extracted from approximately 3 × 10⁵ femoral BM cells. RNA from laser capture microdissected islets was extracted using the miRNeasy Micro Kit (QIAGEN) following the manufacturer's protocol. RNA from cultured islet cells was extracted using the RNeasy Micro Kit (QIAGEN).

Matched serum and CSF samples were obtained from each PSP patient and healthy control. For serum samples, blood was collected from patients into BD Vacutainer SST tubes, left to clot at room temperature and centrifuged at 3000 rpm for 10 min at 4˚C. The serum supernatant was then removed and aliquoted into 1.8 mL aliquots and stored at -80˚C. Minimal red blood cell lysis was checked using a haemoglobin ELISA (ab157707, Abcam) with a threshold of 0.6 g/L [40 (link)]—13 additional samples originally received from the PROSPECT study exceeded this threshold and were excluded from those used in the study. Small RNA was isolated from a 200 µL sub-aliquot of individual serum samples using the miRNeasy Micro kit (Qiagen) with a DNase I treatment (Qiagen).
CSF samples were obtained by lumbar puncture directly into polypropylene collection tubes. Samples were centrifuged at 3000 rpm for 10 min at 4 °C within 1 h of sampling and stored at −80 °C until extraction. CSF samples were checked for any contamination for blood by visual inspection. Small RNA was isolated from a 400 µL sub-aliquot of individual CSF samples using the miRNeasy Micro kit (Qiagen) with a DNase I treatment (Qiagen).

Total RNA was extracted from previously isolated exosomes using the miRNeasy Micro Kit (Qiagen) then concentrated with the RNA Clean & Concentrator-5 (Zymo). Primary WM cells were obtained from bone marrow biopsy samples using CD19+ microbeads selection (Miltenyi Biotec) as previously described and stored at -80°C[27 (link)]. Cells were homogenized with Qiashredder columns (Qiagen) and RNA was extracted from the homogenized lysate using the miRNeasy Micro Kit (Qiagen). The amount of RNA obtained was measured by fluorimetry with the Quant-iT RiboGreen RNA Assay Kit (Thermo Fisher Scientific).

RNA of differentiated MSCs was isolated (miRNeasy Micro Kit, Qiagen, Hilden, Germany) and reversed transcribed (M-MVL RT, Promega, Madison, WI, USA) for quantitative real-time PCR. Genes of interest were detected using specific TaqMan (Thermo Fisher Scientific, Waltham, MA, USA) and IDT (Integrated DNA Technologies, Coralville, IA, USA) probes. Expression levels were evaluated using the 2(-Delta CT) method [4 (link)] with eukaryotic translation elongator factor 2 (Eef2) as internal control.
For RNA sequencing, isolated MSCs were expanded for 4 days and RNA was isolated according to the manufacturer’s protocol (miRNeasy Micro Kit, Qiagen, Hilden, Germany).

Total RNA was isolated from 10 µl of enriched KP-EV (n=37) using the Qiagen miRNeasy micro kit (Qiagen) as per the manufacturer’s instructions. RNA was frozen at -80°C until further use.