Plasma miRNA Extraction and Normalization

miRNAs were extracted from baseline plasma using the Qiagen miRNeasy Serum/Plasma miRNA extraction kit, using adaptations previously described [23] (link). Briefly, 250 µl of plasma was centrifuged at 3000×g for 7.5 minutes at room temperature (RT). Then, 200 µl of supernatant plasma was transferred to a PhaseLock Gel 15 ml tube (5 Prime Inc.) containing 2.0 mL Qiazol (Qiagen), inverted 10 times, and incubated for 5 minutes at RT. After denaturation (but prior to extraction, described below), each sample received a well-mixed, single-use aliquot containing 30 fmol of synthetic C. elegans miRNA (cel-miR-39; Integrated DNA Technologies) and 1 µg of carrier rRNA (Roche Life Sciences, cat# 10 206 938 001) in Qiazol. The sample was inverted 10 times, chloroform (0.2x volume) was added and the sample was inverted again 10 times, and incubated at RT for 2 minutes. The samples were centrifuged at 1500×g for 5 minutes at RT and processed following the remainder of the miRNeasy extraction kit protocol.