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[…] biopsy from shaved skin (0.5 cm2) was taken immediately and stored in RNA later solution (Ambion) at 4°C overnight. Standard procedures performed the RNA extraction, and the integrity was evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies®). Only samples with an Integrity Number greater than 8.0 were considered for microarray procedures. The RNA from three different mice was pooled, and two independents pools were used by each strain (each microarray experiment was performed as biological duplicates). The expression of 14000 genes was analysed using a Mouse Genome 430A 2.0 microarray (Affymetrix®). Background correction (RMA) and quantile normalisation were performed with the Affy package included in Bioconductor of R software. To define significant expression between the biological conditions, a moderate t-test was computed with Limma package implemented in Bioconductor. The biological significance of the altered genes (log⁡FC > 1.5 and adj p value < 0.05) was assayed by a pathway analysis with InnateDb database, David database, and ClusterProfiles module of R using gene ontology (Biological processes), Kegg, and Reactome annotations terms. The microarray data were deposited in the NCBI GEO database (GEOID: GSE99868) and validated by analysing the expression of 7 different genes by RT-qPCR as described below., To validate microarray data, we analysed SFN, FOXN1, Krt15, LHX2, CD34, and SOX9 gene expression by using mouse-predesigned oligos (Assay IDs: Mm.PT.58.42262048.g, Mm.PT.58.13135783, Mm.PT.58.5528981, Mm.PT.58.6480133, Mm.PT.58.8626728, and Mm.PT.58.42739087, resp., IDT®) and K6b-designed oligos (5′-CATCAAATACACCACCAGCG-3′ (forward) and 5′-AAGCAGCCAAAAAGAGAAGC-3′ (reverse)). The quantitative re […]

Pipeline specifications

Software tools affy, limma, DAVID
Databases InnateDB