Transcriptomics,Genomics

Dataset Information

35

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


ABSTRACT: 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 Overall design: 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.

INSTRUMENT(S): [MoEx-1_0-st] Affymetrix Mouse Exon 1.0 ST Array [transcript (gene) version]

SUBMITTER: Paul A Lacaze 

PROVIDER: GSE14534 | GEO | 2009-08-01

SECONDARY ACCESSION(S): PRJNA111615

REPOSITORIES: GEO

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Publications

Combined genome-wide expression profiling and targeted RNA interference in primary mouse macrophages reveals perturbation of transcriptional networks associated with interferon signalling.

Lacaze Paul P   Raza Sobia S   Sing Garwin G   Page David D   Forster Thorsten T   Storm Petter P   Craigon Marie M   Awad Tarif T   Ghazal Peter P   Freeman Tom C TC  

BMC genomics 20090810


Interferons (IFNs) are potent antiviral cytokines capable of reprogramming the macrophage phenotype through the induction of interferon-stimulated genes (ISGs). 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 stimulation with interferon-gamma. Genome-wide changes in transcript abundance caused by siRNA activity were measured using exon-level microarrays in the presence or absence of IFNg  ...[more]

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