GSM4504213: Lexogen_miRXplore_2; synthetic RNA; miRNA-Seq
Blood samples were collected from four healthy volunteers into K2EDTA BD Vacutainer tubes (Beckman Dickinson) and centrifuged within 30 min from collection at 1500 x g for 15 min at room temperature. The plasma fraction was aspirated and transferred into 2 ml tubes and centrifuged again for 15 min at 3000 x g. The supernatant was transferred into new 2 ml tubes and stored at -80 °C until analysis. Total RNA was isolated starting from plasma aliquots of 250 µl using the miRNeasy Serum/Plasma Advanced Kit (Qiagen) according to manufacturer´s instructions. 1 µl of isolation spike-in mix and 1 µl of GlycoBlue Coprecipitant (Invitrogen) were added at the lysis step as described in (Androvic et al., 2019). RNA was eluted into 20 µl of nuclease-free water and stored at -80 °C. RNA eluates were then pooled together to produce standard plasma RNA sample used through the study. Libraries were prepared in technical duplicates starting from 5 µl of plasma RNA pool and 5 µl of miRXplore Universal Reference (2x10^6 copies/µl) according to each manufacturer´s protocol using compatible set of sample indexes. Libraries generated by the same protocol were pooled and run on 5% TBE-PAGE on Mini-PROTEAN tetra cell (BioRad). A region representing fragments with RNA inserts of length 22 nt ± ~10 nt (i.e. fragments originating from miRNAs) was excised from the gel, DNA was eluted into nuclease-free water and purified with SPRIselect reagent (Beckman Coulter).
GEO Accession
GSM4504213