Computational protocol: Regulation of retinal pigment epithelial cell phenotype by Annexin A8

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Protocol publication

[…] Total RNA was extracted from cells following 7 days treatment with DMSO or Fenretinide, using TriZol reagent (Life Technologies) and purified (RNeasy Mini Kit, Qiagen) according to the manufacturers instructions. cDNA was synthesised using Superscript II and E.Coli DNA polymerase according to the manufacturers instructions (Life Technologies), purified (GeneChip® Sample Cleanup Module, Qiagen) and used as a template for the synthesis of biotin-labelled cRNA (GeneChip IVT Labeling Kit (Affymetrix). The biotin-labelled cRNA was purified and fragmented using the GeneChip® Sample Cleanup Module. cRNAs were hybridised to the Human U133A 2.0 Affymetrix Microarray. Data analysis was performed in R using packages from Bioconductor. Data sets for each array were quality assessed using affyQCReport. GenChip Robust Multiarray averaging (GC-RMA) was used to correct background, normalise data and convert fluorescence into expression levels. Differentially expressed genes were identified using a two sample T-test followed by FDR analysis. […]

Pipeline specifications

Software tools affyQCReport, GC–RMA
Application Gene expression microarray analysis
Organisms Homo sapiens
Diseases Retinal Diseases
Chemicals Tretinoin, Fenretinide