Computational protocol: Comparative gene expression profiling between human cultured myotubes and skeletal muscle tissue

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Protocol publication

[…] Functional annotation and classification analysis was also carried out with GeneSpring GX program, running a Gene Ontology (GO) analysis on the gene set selected with the statistical analysis described above. GeneSpring GX computed a p-value to quantify the significance in the GO analysis. This p-value is the probability that a random subset of x genes drawn from the total set of n genes will have y or more entities containing a given GO term. This probability is described by a standard hypergeometric distribution. GeneSpring GX uses the hypergeometric formula from first principles to compute this probability. Since a large number of hypotheses will be tested, some form of correction is required. GeneSpring GX addresses this issue using the Benjamini-Yekutieli correction, which takes into account the dependency among the GO terms. In the GO analysis done in GeneSpring GX, a low p-value therefore implies that the given GO term is enriched. The Gene Ontology data in GX 10.0.2 is compiled from version 1.2 of OBO (Open Biomedical Ontologies).Data were interpreted using Ingenuity Pathways Analysis (IPA) (Ingenuity Systems, Redwood City, CA, USA) http://www.ingenuity.com. The list of significantly regulated genes selected by the microarray analysis described above was loaded in IPA with the following criteria: Reference set: HumanRef-8 v2.0; Direct and Indirect relationships included; filtered by species (human), and in a second round, by tissue (skeletal muscle). Then IPA computed the data to generate significant networks of genes that are associated with particular biological functions, diseases, and molecular processes. It showed also the canonical pathways stimulated by the experiment. IPA used the right-tailed Fisher's exact test to extract significant pathways. IPA canonical pathways are well-characterized metabolic and cell-signaling pathways coming from articles, reviews, books, and KEGG Ligand.Gene cluster analysis was performed with the REEF (REgionally Enriched Features in genomes) program, which scans the genome using a sliding window approach, and calculates the statistical significance of each window using the hypergeometric distribution and the false discovery rate []. […]

Pipeline specifications

Software tools GeneSpring GX, REEF
Databases KEGG LIGAND
Application Epigenome data enrichment
Organisms Homo sapiens
Diseases Mitochondrial Diseases