Mirneasy serum plasma mirna extraction kit

MicroRNAs were extracted from baseline plasma (prior to surgery) using the Qiagen miRNeasy Serum/Plasma miRNA extraction kit, following modifications as previously described (Ng et al, 2009 (link)). Briefly, 250 μl of blood sample was centrifuged at 3000 g for 7.5 min 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, Hilden, Germany), inverted 10 times, and incubated for 5 min at RT. After the denaturation step and prior to extraction (described below), each sample received a well-mixed, single-use aliquot of 30 fmol of synthetic C. elegans miRNA (cel-miR-39; Integrated DNA Technologies, Coralville, IA, USA) and 1 μg of carrier rRNA (Roche Life Sciences, Penzberg, Germany, cat# 10 206 938 001) in Qiazol. The sample was inverted 10 times, chloroform (0.2 × volume) was added and the sample was inverted again 10 times, and incubated at RT for 2 min. The samples were centrifuged at 1500 g for 5 min at RT and processed following the relevant portions of miRNeasy extraction kit protocol.