RNA Extraction from Serum Samples

The extraction of RNA from 200 μL serum samples was performed using the miRNeasy Serum/Plasma Kit (cat. 217184, QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. Synthetic Caenorhabditis elegans (C. elegans) miRNA cel-miR-39 was added to serum samples during the extraction step (validation cohort). This enabled normalization for any nonspecific losses incurred during miRNA purification [18 (link)]. The RNA concentration and purity were assessed using a Nanodrop ND-2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and RNA samples were stored at −80 °C until use. We assessed hemolysis in all serum samples by measuring the absorbance of hemoglobin at 414 nm with a Nanodrop ND-2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and in a randomly selected 50% of the RNA samples by measuring the delta Ct value of miR-23a and miR-451 using qPCR [19 (link)].