Ion and scanning were performed according to standard protocols of the

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Ion and Anti-Spike-RBD mAb scanning were performed according to standard protocols of the manufacturer.Microarray data analysisDye-normalised fluorescent intensities of individual microarray spots were extracted using the Agilent Feature Extraction software 9.5. Data were further normalised by dividing the Cy3/Cy5 ratio of each treatment by the mean Cy3/Cy5 ratio of the controls. Data were then analysed using the TMEV software version 4.3 (http:// [73]. PubMed ID: Genes with significantly altered expression patterns were identified by a modified t-statistic (SAM = significance analysis of microarrays) [74]. Multiple comparison of the complete data set was performed using the lowest possible false discovery rate that allows identification of significantly differentially expressed gene (FDR < 0.03 ). Further descriptive analysis by hierarchical clustering (TMEV 4.3) and principal component analysis (PCA, JMP 8.0, SAS institute; http:// was restricted to the statistically significant genes. Fold changes (FC) of expression levels were calculated using the mean values of each treatment and the mean of the respective controls. A complete list of FC-values of all significantly differentially expressed genes is included in the supplementary information section of this paper (Additional file 1). The microarray data have been submitted to the Gene Expression Omnibus (GEO) database (series no. GSE16727, http://www.ncbi. set enrichment and pathway analysispredefined gene sets are provided by the Molecular Signatures Database (MSigDB) and include five different types of databases (C1 to C5). For our analyses we used the databases C2 (gene sets collected from various sources such as online pathway databases, publications in PubMed including microarray studies, and knowledge of domain experts), C3 (transcription factor targets, i.e. genes that share a transcription factor binding site defined in the TRANSFAC database version 7.4, http:// and C5 (gene sets of the Gene Ontology (GO) database, Further details are explained on the MSigDB homepage jsp. Since GSEA does not allow the analysis of multiple datasets, analysis was performed pair wise comparing each treatment with the control. Furthermore, pathway analysis was performed by means of the Database for Annotation, Visualisation and Integrated Discovery (DAVID) [36] (http://david.abcc. the list of differentially expressed genes identified by SAM (see above).RT-PCRIn order to identify biological pathways and functions associated with the changes in gene expression patterns, transcription profiles were analysed by Gene Set Enrichment Analysis (GSEA) [34,35]( edu/gsea/). GSEA is based on ranking of the genes according to their statistical significance and comparison of the patterns to sets of predefined genes. ThesecDNA was synthesised from RNA using the RevAidTM First Strand cDNA Synthesis Kit (MBI Fermentas, St. Leon-Rot, Germany) according to the manufacturer's instructions. Primers were designed using the computer program Primer3 [75] or Beacon Designer 7 (Premier Biosoft, Palo Alto, USA; (http://www.PremierBiosoft. com) and purchased from Invitrogen. Primer sequences are listed in Table 4. Target genes and the reference gene RPL41 [76] were amplified from 1 l of cDNA using 1 unit of Taq Polymerase (Promega, Mannheim,.