Computational protocol: Differential effects of class I isoform histone deacetylase depletion and enzymatic inhibition by belinostat or valproic acid in HeLa cells

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Protocol publication

[…] RNA integrity was quality checked on the Agilent 2100 Bioanalyser (Agilent Technologies, Santa Clara, CA), then processed and hybridized onto Affymetrix arrays according to the manufacturer's protocol. Briefly, 5 μg RNA pr sample was used to to generate biotin-labeled antisense cRNA. After fragmentation, the labeled cRNA samples were hybridized to Affymetrix HG-U133 Plus 2.0 arrays (Affymetrix, Santa Clara, CA), washed and stained with phycoerytrin conjugated streptavidin, and finally scanned in the Affymetrix GeneArray® scanner to generate fluorescent images, as described in the Affymetrix GeneChip® protocol. All statistical analyses, including pre-processing of data was carried out in R, version 2.3 (R development core team. R: A language and Environment for Statistical Computing; ). DNA chips were checked for quality assurance parameters such as visual image inspection, replicate scatter plots and RNA degradation plots, before normalization for mean overall expression using the gcrma package (GC robust multiarray algorithm) []. Agglomerative hierachical clustering showed that the biological replicates clustered together as expected (data not shown). The statistical linear model based method of Limma (linear models for microarray data package) [] was found to be most sensitive at identifying genes with differential expression between control and each condition. Raw p-values were adjusted for multiple testing using the Benjamini & Hochberg method [] to reduce the number of false positives, and a 5% significance threshold applied. Comparisons of gene lists between conditions was performed using VennMapper []. […]

Pipeline specifications

Software tools GC–RMA, limma
Application Gene expression microarray analysis
Organisms Homo sapiens
Diseases Neoplasms
Chemicals Valproic Acid