Isolation and Analysis of Blood miRNAs

RNA isolation was performed as described in detail earlier [13 (link), 14 (link), 15 ]. In short, 5 mL blood was centrifuged at 5000 g for 10 min at room temperature. Total RNA including miRNA from the cell pellet was isolated using the miRNeasy kit (Qiagen, Hilden, Germany) according to the manufacturer’s instruction. RNA integrity was evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). Only samples with an RNA integrity number of at least seven were used for further analyses. The total RNA prepared from individual samples was analysed at the Genomics Core Facility of the German Cancer Research Center (DKFZ) in Heidelberg on the Agilent Human miRNA Microarray Release 21.0 according to the detailed manufacturer’s protocol that is available from Agilent and includes process controls. Briefly, fluorescently labelled miRNA was prepared according to the Agilent miRNA Complete Labelling and Hyb Kit protocol. Hybridisation was performed in a SureHyb chamber (Agilent Technologies) at 55 °C for 20 h. Microarrays were scanned using the Agilent Scanner G2505C. After quantile normalisation, fold changes were calculated based on mean values of technical replicates. In total, 2549 human miRNAs were analysed for differences in their blood levels.