Unisp5

Total RNA, including miRNA, was purified from 250 μl cell-free CSF and 200 μl serum samples using the miRNeasy Serum/Plasma Kit (Qiagen, P/N 217184). The protocol was applied according to the manufacturer’s recommendations with the following modifications. QIAzol Lysis Reagent mixture containing 1.25 μg/ml MS2 bacteriophage RNA (Roche Applied Science) and 1 μl RNA spike-in template mixture containing synthetic UniSp2, UniSp4, and UniSp5 (Exiqon, P/N 203203) was added to all samples. The total RNA was not treated with DNase and eluted with 22 μl RNAse-free water and stored at -80°C until use.

Total RNA was extracted from 200 µl of maternal serum using the miRCURY RNA Isolation Kit—Biofluid (Exiqon, Denmark) following the manufacturers’ protocol, with inclusion of an on column DNAse digestion step. For increased reproducibility, a carrier RNA (1 µg MS2 phage RNA; Roche, UK)) was added prior to extraction along with RNA spike-in controls (UniSp2, UniSp4, UniSp5 Exiqon, cat no. 203203) to monitor the technical quality of the RNA isolation, cDNA synthesis and the presence of PCR inhibitors. Samples contaminated by haemolysis where ΔCt miR-23a-3p–miR-451a was > 7 were excluded. Standard methods of RNA yield/purity are inaccurate due to carrier RNA, however we adhered to best practise guidelines by standardising input amounts based on isolating from identical starting volumes and using the same volume of purified RNA for all downstream processes [32 (link)].

Eleven additional randomly chosen mLi and 11 pCRC tissue samples were selected for qPCR validation of increased expression of Mir-210_3p in mLi compared to pCRC. Synthetic RNA Spike-Ins UniSp2, UniSp4 and UniSp5 (Qiagen, Düsseldorf, Germany Cat. No. 339390) were added pre-isolation. RT-PCR was done with miRCURY LNA RT Kit (Qiagen Cat. No. 339340), adding UniSp6 and cel-miR-39–3p RNA Spike-Ins (Qiagen Cat. No 339390). qPCR was done using Qiagen miRCURY SYBR Green Kit (Qiagen Cat. No. 339345) and miRCURY LNA miRNA PCR Assays (Qiagen Cat. No. 339306). Mir-103 (Qiagen primer Cat. No. YP00204306) was used as reference miRNA, while the Mir-210_3p primer was Qiagen Cat. No. YP00204333. Two PCR replicates were run per primer assay. Mir-210_3p Cq values were normalized to the reference miRNA to obtain the dCq value. Welch two-sided t-test was used to compare the dCq values, to test if the expression levels were different between mLi and pCRC. (Supplementary File S5).

EVs were purified by centrifugation at 100,000× g for 1.5 h at 4 °C. The pellet was lysed with 1000 µL Qiazol, supplemented with 1 µL of a synthetic RNA mixture containing three different synthetic control RNAs (UniSp2, two fmol/mL; UniSp4, 0.02 fmol/mL; UniSp5, 0.0002 fmol/mL; Qiagen, Hilden, Germany). RNA extraction was performed using 200 µL chloroform, and phase separation was achieved by centrifugation for 15 min at 12,000× g at 4 °C. RNA was extracted from the upper aqueous phase and purified on a QIAcube liquid handling robot using the miRNeasy Mini kit (Qiagen) with the following modifications: glycogen (Ambion, Austin, TX, USA) was added to the aqueous phase to a final concentration of 50 mg/mL and precipitated with 750 µL 100% ethanol. Columns were washed twice with RPE buffer and RNA was eluted in a single round in 30 µL nuclease-free water and stored at –80 °C.
Small RNA Libraries were prepared from 2 µL RNA, using the CleanTag Small RNA Library Prep Kit (TriLink Biotechnologies, San Diego, CA, USA) according to the manufacturer’s protocol. cDNA libraries were amplified in 21 PCR cycles. Equimolar amounts were pooled and libraries underwent 50 cycles of single-end sequencing on a HighSeq 2500 (Illumina, San Diego, USA).