Labelled cDNA was hybridized on the microarrays, which were subsequently washed, stained and scanned. Quality control and statistical data analysis Data was analysed with bioconductor (R version 2.10.0; http://www.bioconductor.org) packages affy [22], gcrma [23] and limma [24]. Quality control of the microarray consisted of visual inspection of various diagnostic plots, namely boxplots Selleck BIRB 796 of transcript intensities, image plots of arrays and MA plots of raw data. Additionally, parameters from the Affymetrix software were evaluated. Moreover, RLE (Relative Log Expression) and NUSE (Normalized Unscaled Standard Error) plots were constructed [25]. Of 38 analyzed
arrays, one did not meet the quality requirements and was therefore excluded from further analysis. Data pre-processing
and expression value calculation were carried out using two procedures, yielding 2 separate datasets. In the first, a combination of rma convolution method for background adjustment [26], invariantset for normalization [27], pm correction as from the mas manual, and liwong method summarization [27, 28] were applied. In the second procedure, all the pre-processing steps were performed simultaneously using gcrma [23]. In order to find differentially expressed genes a statistical model was formulated (p < 0.05) to compare gene expression in bacteria exposed to fosfomycin concentrations Selleckchem Volasertib c1 and c4 with that of the control (c0) at a given time point. To decrease false discovery rate, the results coming from different pre-processing procedures were combined and only the intersection of genes, differentially expressed following both procedures were taken into account for the biological interpretation of the results [29]. Pathway analysis Biochemical reactions from S. aureus metabolic network reconstruction iSB619 [4] were obtained from BIGG database http://bigg.ucsd.edu/ and coupled with TIGR S. aureus annotation [30] downloaded from TIGR CMR database http://cmr.tigr.org/tigr-scripts/CMR/CmrHomePage.cgi. Pathway
database and expression profiles for all experimental time points were imported to Pathway Studio software (version 4.0; Ariadne Genomics Inc). tuclazepam Differentially expressed genes were queried for presence in metabolic network. Pathways constructed in Pathway Studio were examined and interpreted manually. Pathway Studio .gpc file is available as P5091 nmr Additional file 2. Gene set enrichment analysis (GSEA) [31] was applied to search for groups of genes involved in the same processes (gene sets) that were altered significantly by fosfomycin treatment. Individual GSEA was performed for a data set including control and both fosfomycin treatment concentrations (1 and 4 μg/ml) for the selected time point. Gcrma-normalized data was filtered for signal intensity greater than 10. The signal intensities from the same time point were overlapped on 40 gene sets (see Additional file 3) based on TIGR S.