Combined genome-wide expression profiling and targeted RNA interference in primary mouse macrophages

Here we have used targeted RNA interference to suppress the expression of a number of key genes associated with IFN signalling in murine macrophages prior to activation. Genome-wide changes in transcript abundance caused by siRNA activity were measured using Affymetrix exon-arrays in the presence or absence of IFNγ stimulation. Transfection of murine bone-marrow derived macrophages (BMDMs) with non-targeting (control) siRNA and 11 sequence-specific siRNAs was performed using a cationic lipid transfection reagent (Lipofectamine2000) prior to stimulation with IFNγ. The 11 genes targeted were Ifnb1, Irf3, Stat1, Stat2, Nkfb2, Irf5, Casp4, Ifi47, Lyn, Sod2, and Traf1. Total RNA was harvested from cells and gene expression measured on Affymetrix GeneChip Mouse Exon 1.0 ST Arrays. Keywords: RNAi, stress response, macrophage, IFN, interferon, siRNA, lipofectamine, cytokine Transfection of murine bone-marrow derived macrophages (BMDMs) with non-targeting (control) siRNA and 11 sequence-specific siRNAs was performed using a cationic lipid transfection reagent (Lipofectamine2000) prior to stimulation with IFNγ. Total RNA was harvested from cells and gene expression measured on Affymetrix GeneChip Mouse Exon 1.0 ST Arrays. 90 samples were analyzed in total. 1. Control experiments: 18 arrays studying the host response to Lipfectamine2000 and RiscFree non-targeting control siRNA (ThermoFisher) at 5 and 24 hours post treatment. 2. siRNA targeting experiments: 72 arrays in 12 different siRNA treatment groups, with each treatment group containing 6 arrays (3 replicates unstimulated, 3 replicates stimulated with IFNg). In each group a different targeting siRNA was used and in one group control non-targeting siRNA (RiscFree siRNA ThermoFisher) was used.