Serum microRNA Extraction and Validation

Extraction of RNA from serum samples (200 μL) 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 enables normalization for any nonspecific losses incurred during miRNA purification [32 (link)].The RNA concentration and purity were assessed using a Nanodrop ND-2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). 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 later in 50% randomly selected RNA samples by measuring the delta Ct value of miR-23a and miR-451 by RT-qPCR [33 (link)].