Kristiansen et al. BMC Genomics 2011, 12:551 http://www.biomedcentral.com/1471-2164/12/551 Page 4 of 17 Figure 2 Schematic overview of gene-level analysis. Rat sympathetic neurons were cultured in vitro for 6 days and then maintained in 1) medium containing NGF, or 2) medium without NGF supplemented with anti-NGF antibody or 3) medium without NGF containing anti-NGF antibody and the MLK inhibitor CEP-11004 at 400 nM. Three independent experiments were performed. After 16 hours, RNA was isolated and processed and gene expression analysed using Affymetrix rat exon microarrays. The summarisation algorithm, PLIER (Probe Logarithmic Intensity Error) was used to compute gene-level expression values. At the gene level, we identified 1228 differentially expressed genes with a Limma FDR (false discovery rate) adjusted p-value of < 0.05. This number of differentially expressed genes was reduced to 48 up-regulated and 86 down- regulated genes when applying a more stringent statistical test (FDR adjusted p-value < 0.01) and an absolute fold change threshold of > 2. A - see additional file 1 for a list of these genes. B - see additional file 2 for a list of these genes. expression between each of the three different treatment groups (+NGF, -NGF, -NGF+CEP-11004). Each indivi- dual comparison generated a list of differentially expressed genes which were either up- or down-regu- lated in sympathetic neurons. When comparing the +NGF and -NGF treatment groups this analysis revealed 415 genes that were up-regulated and 813 genes that were down-regulated (Figure 2). A more stringent statis- tical threshold with an FDR-adjusted p-value of < 0.01 reduced this number to 164 and 379 up- and down- regulated genes respectively. Further analysis revealed that of the up-regulated genes with a FDR adjusted p- value of < 0.01, 48 genes had a fold change of greater than 2 (Additional file 1). Similarly, the expression of 86 of the genes that were down-regulated changed in level by greater than 2-fold (Additional file 2). We also checked our microarray data for the genes previously shown to be regulated by NGF withdrawal in sympathetic neurons, such as c-jun, dp5, bim, egln3 and cyclinD1 and found that their expression had changed as predicted (Table 1; [13-17,24,31-35]). Importantly, the induction after NGF withdrawal of those genes pre- viously defined as targets of the MLK-JNK-c-Jun path- way, c-jun, bim, dp5, mkp1 [17,19,24,26,34] was reduced by CEP-11004. Gene Ontology analysis and unsupervised hierarchical clustering Gene Ontology covers three domains: cellular component, molecular function and biological process. However, to obtain an overview of the functional categories and the biological relevance of the genes regulated by NGF with- drawal, we used an alternative strategy of functional analy- sis (rather than the standard Gene Ontology analysis). Functional enrichment analysis of Gene Ontology terms using DAVID identified those annotations that were sig- nificantly overrepresented amongst the list of genes signifi- cantly de-regulated after NGF withdrawal compared to a reference gene list consisting of all of the annotated genes represented on the microarray. All genes significantly up- or down-regulated (FDR = 0.05) after NGF withdrawal (1228 in total) were eligible for this analysis. Gene lists that contained up- or down-regulated genes were analysed separately. Following NGF withdrawal, major functional categories that were especially up-regulated included intracellular signalling cascade, ion transport and tran- scription (Figure 3). In contrast, the down-regulated genes appeared to be involved in cell cycle and intracellular transport as well as nucleoside and ion binding. Interest- ingly, twice as many genes associated with the cell death process were down-regulated compared to up-regulated.