Identification of miR-146a target genes in mouse monocyte subsets. Mus musculus

To gain insight into the mechanisms underlying miR-146a-mediated modulation of Ly6Chigh monocyte function, we compared the expression profiles of Ly6Chigh and Ly6Clow monocytes in miR-146a+/+ (WT) versus miR-146a-/- (KO) conditions. Overall design: Ly6Clow and Ly6Chigh monocyte subsets were isolated from spleen samples of miR-146a-/- mice or control littermates using a FACSAria sorter, and total RNA of each cell subsets were extracted using miRNeasy mini kit (Qiagen). The integrity of isolated and pooled RNA was assessed for each sample using an Agilent 2100 Bioanalyzer (Agilent, Waldbronn, Germany) and amount was checked with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). The generation of biotinylated anti-sense cRNA was performed with 100 ng of total RNA using the Affymetrix IVT Express protocol (3' IVT Express 2010 technical manual P/N 702646 Rev8). Following fragmentation, 15 µg of the cRNA were hybridized for 16 h at 45 °C on GeneChip Mouse 430_2 arrays in the Affymetrix oven 645. GeneChips were washed and stained in the Affymetrix Fluidic station 450 with HWS kit. Chips were scanned using the Affymetrix GeneChip scanner 3000 7G, and the raw data were generated with the Affymetrix Expression Console software v 1.2.1. Raw data were analyzed with High Performance Chip Data Analysis (HPCDA) and database BioRetis using one vs. one chip analyses.