Isopropanol

Trizol LS was used for RNA extraction from 400 μl plasma (Ambion, Life Technologies) as described previously (Papadaki et al., 2018 (link)). Briefly, following denaturation by Trizol LS, 25 fmoles of the synthetic C. elegans miRNA, cel-miR-39 (Qiagen GmbH, Hilden, Germany) were added in each sample to serve as an exogenous control. Chloroform was added for phase separation and after centrifugation, an equal volume of 700 μl of aqueous phase from each sample was precipitated by adding 0.7 volumes of isopropanol and 1 μl of glycogen (Qiagen). RNA pellet was resuspended in 50 μl RNAse-free water. RNA from all samples was kept at −80°C until further use in the subsequent cDNA synthesis step.

Lysate aliquots (2–3 × 100 µL per sample) from the same sample were pooled, and if required RLT or QIAzol was added, to give a final lysis volume of 300 µL. Samples were transferred to 2 ml PhaseLock tubes (QuantaBio). For RLT lysates specifically, a half volume of phenol:chloroform:isoamyl alcohol (25:24:1 v:v:v; Sigma) was added before brief shaking and centrifuging at 4 °C. For both RLT and QIAzol samples, a half volume of chloroform:isoamyl alcohol (24:1 v:v) was added before shaking, a 3 min RT incubation and centrifugation at 4 °C. The aqueous phase was then transferred to a 1.5 ml Eppendorf tube and mixed with a 1.5 volume of isopropanol (Sigma). After thorough pipette mixing, the isopropanol mixture was applied to an RNeasy MinElute spin column and total RNA was extracted using the miRNeasy Micro Kit (Qiagen) with a DNase treatment. Samples were eluted in 14 µl nuclease-free water. RNA samples were assessed both quantitatively and qualitatively using the High Sensitivity Total RNA 15nt Analysis DNF-472 Kit on a 48-channel Fragment Analyser (Agilent). Total RNA yield was 0.31 ± 0.11 ng (mean ± SD) in the RLT lysate samples and 1.45 ± 0.51 ng in the QIAzol lysate samples; RNA integrity could often not be computed due to low input.

Total genomic DNA was either extracted using the QIAamp^® DNA Mini Kit (Qiagen) or by classical isopropanol precipitation as follows: the cell pellet was incubated with 200 μl lysis buffer (0.2 mg/ml Proteinase K, 0.2% SDS, 5 mM EDTA in DPBS) for 3 h at 50°C. Afterwards 20 μl of 3M sodium acetate pH 5.2 and 300 μl of isopropanol (VWR Chemicals) were added to the lysates followed by incubation for 20min at −20°C and centrifugation (13000xg) for 30min at 4°C. Pellets were washed twice in 70% Ethanol (Honeywell) and resuspended in H₂O (Braun). The total genomic DNA was quantified using NanoDrop 2000 Spectrometer (Thermo Scientific).