Serum microRNA Extraction and Profiling

For all analyses, RNA was extracted from 200 μL of serum using the miRNeasy serum kit (Qiagen) into a final volume of 100 μL nuclease free water as described previously (12 (link)). All isolated RNA was stored at -80°C. RNA yield was maximised with the addition of MS2 carrier RNA (Roche, final concentration 1.25 μg ml⁻¹) to Qiazol prior to isolation and the exogenous spike-in miRNA cel-miR-39-3p (5.6 × 10⁸ copies) was used as an initial quality control for extraction efficiency. All samples underwent quality control analysis prior to subsequent target miRNA qRT-PCR analyses (12 (link), 16 (link)). Quality control analysis included quantification of cel-miR-39-3p, the endogenous housekeeper miR-30b-5p and the haemolysis control miRNAs miR-451a and miR-23a-3p as previously described (12 (link)). Consistency of extraction was acceptable for all samples analysed (14 (link)). Haemolysis assessment was performed by calculating the delta Ct values for miR-23a-3p minus miR-451a, detailed in (14 (link)).