Fibroblast cell line from individuals affected by Mucolipidosis type IV (Cy5 labelled) versus fibroblast cell line from normal control individuals (Cy3 labelled). RNA samples extracted from different subjects were pooled into a single RNA mix. Each pair of TIFF image files was processed using GenePix Pro 4.1 (Axon Instruments). These files were firstly examined for quality control: spots showing no signal or obvious defects were accordingly flagged by GenePix Pro and excluded from analysis; besides, all �negative� control spots were manually flagged and excluded from further analysis. All statistical and graphical analysis were carried out in the R computing environment using the Linear models for Microarray data package (Smyth, 2004) from Bioconductor project (www.bioconductor.org) (Gentleman et al., 2004). For each microarray it was performed local background subtraction followed by global lowess normalization within each slide followed by scale normalization between slides. The two replicate spots per gene in each array were used to maximize the robustness of statistical analysis on each gene via the �lmFit� function within Limma (Smyth, 2004). Differential expression of genes was determined using an empirical Bayes (EB) approach within Limma (Smyth, 2004). After EB analysis, genes were ranked as being differentially expressed in decreasing order of the B-statistic and p-value.
Data processing
Global lowess normalization within and scale normalization between arrays by Limma package
