Computational protocol: Identification of p38 MAPK as a novel therapeutic target for Friedreich’s ataxia

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Protocol publication

[…] GM3816 fibroblasts were transfected in triplicate with clone gFA11 or clone Mut1 four times over two weeks and grown in BHB-based medium after the first transfection. RNA was extracted at day 14 by using the Qiagen mRNAeasy kit. RNA quality control, reverse transcription, and hybridization to the Human Gene 2.0 ST GeneChips (Affymetrix Inc., Santa Clara CA) were performed by the University of Pennsylvania Molecular Profiling Facility. To quantify expression levels of targeted genes, RMA normalization was applied using Partek Genomics Suite v6.6, yielding log2-transformed normalized intensities. Genes were analyzed for differential expression using Significance Analysis of Microarrays (SAM; samr v2.0). The gene list was then sorted by q values, a statistical parameter that takes into account the magnitude of the differences, the precision of the data, and correction for multiple comparisons. The q value calculated by SAM represents false discovery rate on a scale of 0–100.The 301 genes with a q value of zero were analyzed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.7. Functional annotation analysis finds the most overrepresented biological terms associated with a given gene list; the annotation coverage includes more than 40 categories, including GO, KEGG, and BioCarta pathways. An enrichment score (ES) >1.3 is considered significant. Genes that differed in expression >1.5 fold and were associated with a q value ≤10 were also analyzed through the use of Ingenuity Pathway Analysis (Ingenuity® Systems, www.ingenuity.com) through the UPenn Molecular Profiling Facility. Microarray data are available in the ArrayExpress database (www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-4699. […]

Pipeline specifications

Software tools Partek Genomics Suite, SAM, IPA
Databases ArrayExpress BioCarta
Application Gene expression microarray analysis
Diseases Friedreich Ataxia, Heredodegenerative Disorders, Nervous System