Microarray experiment and data processing

Cy3-labeled cRNA was synthesized using the one color Quick Amp Labeling kit (Agilent Technologies) according to the manufacturer’s instructions. cRNA fragmentation and hybridization onto the Whole Human Genome Oligo Microarray V2 was carried out as recommended by the Agilent One-Color Microarray-Based Gene Expression Analysis Protocol V5.7. Data were analysed using the R software and several packages from BioConductor [43] and other sources. Preprocessing steps comprised background correction (“normexp”), quantile normalisation, probe summarisation, and log2 transformation. For the background correction, fitted intensities were calculated by the convolution of normal and exponential distributions to observed foreground and background intensities [44]. For a robust analysis, median values were calculated from replicate samples for each gene. Differentially expressed genes (DEG) were obtained by selecting genes with a minimal fold change of two and significant changes to untreated controls keeping false-discovery rate adjusted p values of 10 % according to the rank products method in order to apply robust statistics for small replicate sizes [45, 46]. In order to further functionally characterise the selected genes, GOstats for identifying gene ontology terms was applied [47]. A functional analysis of the selected gene list was done with SPIA, the signaling pathway impact analysis [48]. GSEA served for understanding the genome-wide expression of all genes [49] via cellHTS2 [50]. The data sets are available under GEO accession number {"type":"entrez-geo","attrs":{"text":"GSE61356","term_id":"61356"}}GSE61356.