Unisp6 rna spike in

In an initial approach, we evaluated Cq values of the synthetic UniSp2 and UniSp6 RNA spike-in (Exiqon) and the miRNAs hsa-miR-103a-3p and hsa-miR-451a in four plasma-derived RNA samples, isolated with Q and E modified protocols, in presence of yeast RNA (yQ and yE) or MS2 bacteriophage RNA (mQ and mE) as carrier and also without RNA carrier (wQ and wE) (Fig 1). We used hsa-miR-103a-3p, a well-known tissue and plasma endogenous normalizer [29 (link),30 (link)] and hsa-miR-451a, a marker of hemolysis [31 (link)]. After the analysis of preliminary results we studied more miRNAs (see next section) and compared the Cq values obtained with the different RNA isolation protocols used.

CSF samples (0.5 ml) of cohorts 1 and 2 were spiked with 1 μg MS2 carrier RNA (Roche Applied Science) to increase the yield of RNA isolation [23 (link)]. Liquid was removed by freeze-drying on an Alpha 1–2 LDplys freeze dryer (Christ, Osterode am Harz, Germany) and samples were resuspended with 200 μl RNase-free water. RNA was isolated using the miRCURY RNA Isolation kit for biofluids (Exiqon, Vedbaek, Denmark). Equivalent CSF volumes were used as input for the RNA isolation. The expression profiles of miR-24 and miR-16 have been reported to be stable in several bodily fluids and tissues [24 (link)–27 ], and were therefore used for normalization purposes.
RNA isolation of cohort 3 samples was performed on 300 μl CSF again using the miRCURY RNA Isolation kit for biofluids (Exiqon, Vedbaek, Denmark) according to the manufacturer’s protocol and including the on-column DNase treatment. To optimize RNA yield per sample, 2 μg glycogen carrier was added to the lysis solution. Secondly, to monitor RNA isolation and proper reference miRNA normalization, per sample, 150 pmol synthetic UniSP6 RNA spike-in (Exiqon) was added to the lysis solution. Eluted RNA was directly stored at − 80 °C.