Unknown,Transcriptomics,Genomics,Proteomics

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Effect of scarification on gene expression in the skin of C57BL/6 mice

ABSTRACT: To study the effects of scarification on gene expression in the skin we performed microarray analysis on intact skin (control) and skin collected 2 h after scarification. Comparison of microarray data between the two groups showed that 451 probe sets (1.0% of a total 45038 probe sets on the arrays) representing 338 known genes were upregulated ≥ 2.0 fold in scarified skin samples when compared to controls. Further analyses of these genes showed that many were associated with an inflammatory immune response. Others were associated with wound-repair, response to stress, apoptosis and transcriptional regulation. The induction of high levels of genes encoding the neutrophil chemoattractants Cxcl1 and Cxcl2, the G-CSF receptor Csf3r, the GM-CSF receptor Csf2rb, the calcium binding proteins S100a8 and a9 (S100a8 and S100a9, respectively) the cytokines Ilb and Il6, and Cd14, Mmp9 and Sell, amongst many others, suggested a significant influx of neutrophillic granulocytes into the skin after scarification. Indeed, the expression of Cxcl2 and Il1b were up-regulated 50 fold in scarified skin when compared to intact skin. C57BL/6 mice (6-12 weeks old), 2-3 per group used. Prior to scarification approximately 1 cm2 area of hair covering the site of scarification was trimmed using curved scissors and then removed completely with an electric razor. Twenty-four hours later a 23-gauge needle was used to create a 5 mm long abrasion in the epidermal layers of the skin at the scarification site. 2 h later, ear skin was collected into RNAlater (Ambion, location) and stored at -80 oC prior to analysis. The quality of RNA for microarray analysis was first determined on an Agilent BioAnalyzer 2100 (Agilent Technologies). RNA was reverse transcribed to cDNA, amplified and labelled using the Genechip 3’ IVT Express Labelling Kit (Affymetrix, Santa Clara, CA, USA) according to the manufacturer’s instructions. Samples were hybridized to HT_MG-430_PM Affymetrix chips (Affymetrix), stained and scanned by ARK Genomics (ARK Genomics, Edinburgh, UK).

ORGANISM(S): Mus musculus

SUBMITTER: Neil Mabbott

PROVIDER: E-GEOD-34955 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Langerhans cells (LCs) are antigen presenting cells residing in the epidermis. Due to the difficulties with obtaining sufficient quantities of LCs for functional studies, many controversies exist about their origin and function. To gain insights into the molecular mechanisms underpinning LC biology and to elucidate how similar they are to a classical tissue resident DCs of myeloid origin, we undertook a whole transcriptome analysis of human skin migratory CD1a+ LCs and CD11c+ DDCs, stimulated with an epidermal pro-inflammatory cytokine, TNF-α over a time course of 24h. RNA was extracted from 250,000 human skin migratory CD1a+ LCs and CD11c+ DDCs stimulated in culture with 25ng/ml of TNF-α for 0, 2, 8 and 24h. RNA concentration and integrity was determined with an Agilent Bioanalyser (RIN of 7.0 or above) and gene expression analysis was carried out using the Human Genome U-219 Affymetrix platform by ARK-Genomics Centre, The Roslin Institute, Edinburgh. Expression data were normalised using the Robust Multichip Average (RMA) package. 2,334 transcripts regulated by exposure of skin DCs to TNF-α were identified with Bayesian Estimation of Temporal Regulation, a cut-off threshold 0.05 , for genes showing ≥ 1.5 fold difference between the maximum gene expression level and time 0 control in log2(x) RMA normalised gene expression. The interactive 3D diagram presenting the transcriptomic networks in human skin DCs can be viewed at: http://www.macrophages.com/LC_vs_DC RNA was extracted from 250,000 human skin migratory CD1a+ LCs and DDCs stimulated in culture with 25ng/ml of TNF-α for 0, 2, 8 and 24h. RNA concentration and integrity was determined with an Agilent Bioanalyser (RIN of 7.0 or above) and gene expression analysis was carried out using the Human Genome U-219 Affymetrix platform by ARK-Genomics Centre, The Roslin Institute, Edinburgh. Expression data were normalised using the Robust Multichip Average (RMA) package. 2,334 transcripts regulated by exposure of skin DCs to TNF-α were identified with Bayesian Estimation of Temporal Regulation, a cut-off threshold 0.05 , for genes showing ≥ 1.5 fold difference between the maximum gene expression level and time 0 control in log2(x) RMA normalised gene expression. *submitter cannot locate the CEL files

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Effect of scarification on gene expression in the skin of C57BL/6 mice

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