Mirneasy serum plasma advanced

For this study, whole blood from 27 non-small cell lung cancer patients (NSCLC) was collected at the University Medical Center Hamburg-Eppendorf. Blood was collected before, and during, the treatment of patients. All patients gave written informed consent for retention and analysis of their blood for research purposes (local ethical review board of the Ärztekammer Hamburg, approval PV5392). An overview of patients’ characteristics is given in Table 2. Similarly, 20 healthy donors (age 40–65 years) were enrolled after giving written informed consent at Clinical Research Services GmbH (CRS) Wuppertal (local ethical review board of the medical association Nordrhein, ref no. A 18/009). Based on the results of the previous miRNA ring trial [10 (link)], the two best performing kits, miRNeasy Serum/Plasma Advanced and ExoRNeasy (both QIAGEN), were used for isolation of cell-free, and EV-associated miRNA from plasma, respectively. Next, systematic comparison of different NGS-based small RNA sequencing and microarray platforms was designed as a multicentric ring study (herein after referred to as screening study). Validation of the identified miRNA candidates was performed using a two-tailed RT-qPCR [17 (link)] and a customized miRCURY LNA miRNA assay (QIAGEN) (herein after referred to as validation study).

RNA was extracted from either fresh or frozen plasma according to the manufacturer’s instructions using 6 commercially available kits; Kit A: miRNeasy Serum/Plasma (Qiagen, Germany), Kit B: miRNeasy Serum/Plasma Advanced (Qiagen, Germany), Kit C: Quick-cfRNA Serum & Plasma (Zymo Research, USA), Kit D: Isolate II miRNA (Bioline, Australia), Kit E: PureLink RNA Mini kit (Invitrogen, USA), Kit F: Monarch Total RNA Miniprep kit (New England BioLabs, USA). All extractions were performed in duplicate for each animal, except for the PureLink and Monarch kits due to sample and reagent availability. Following extraction and elution of RNA, samples were immediately frozen at −80 °C.