Martin et al. BMC Genomics 2010, 11:418 http://www.biomedcentral.com/1471-2164/11/418 Page 3 of 20 h s i f d e f n i n e g o h t a p o t e s n o p s e r e m o t p i r c s n a r T Fed PFL RNA from 6 groups of 4 fish Infected (fed) AFL RNA from 6 groups of 4 fish Transcriptome responses to starvation Pooled Reference RNA Starved PSL RNA from 6 groups of 4 fish Infected (starved) ASL RNA from 6 groups of 4 fish h s i f d e v r a t s n i n e g o h t a p o t e s n o p s e r e m o t p i r c s n a r T Effect of prior feeding on response to infection Figure 1 Experimental design and hybridizations. Diagram outlining the experimental setup and hybridization strategy for microarray analysis. For each experimental group 6 pools of liver RNA extracted from 4 individuals was used. Experimental groups were: PBS control/fed liver (PFL), from fish fed a normal diet and injected with PBS: PBS control/starved liver (PSL), fish starved for 28 days and injected with PBS: Aeromonas salmonicida infected/fed liver (AFL), fish fed normal diet and injected with 100 μL of bacteria (A. salmonicida, 109 CFU ml-1); A. salmonicida infected/starved liver (ASL) fish starved for 28 days and injected with 100 μL of bacteria (A. salmonicida, 109 CFU ml-1). All fish were sampled 24 h following injection. The pooled control was an equal mixture of RNA pools from each group. All hybridizations were between experimental groups and the pooled control. For each group of 6, 12 hybridizations were performed which included a dye swap. significantly affected by these treatments. The numbers of genes differentially expressed are shown in Fig. 2. For annotation of the microarray, 76% of the genes could be assigned to a functional protein and 46% of the genes had a gene ontology (GO) identifier. This allowed statis- tical analysis for enrichment for GO biological processes to help interpret the changes in the transcriptome fol- lowing starvation, infection and the effect of feeding regime on the response to infection. Transcriptome changes following starvation Of 959 genes significantly altered by starvation, 288 were altered by greater than 2 fold, with 109 showing an increased and 179 a decreased expression as a result of starvation (Table 1 & Additional file 1 Table S1). To further interpret this extensive list of genes, enrichment for GO biological process was performed. Seventy one biological processes were found to be significantly altered following starvation. The GO enrichments indi- cated major alterations in biological processes related to fatty acid and steroid metabolism, energy metabolism, cell structure, immune status, protein metabolism and ion transport (Table 2, Additional file 2 Table S2). Genes encoding proteins related to a number of key biological processes that were altered in expression following starvation are outlined below. a. Up regulated genes b. Down regulated genes AFL (Au) 145 (ACu) 29 AFL (Ad) 25 (ACd) 36 PSL (Cu) (ABu) 287 (ABCu) 31 49 (ABd) 100 (ABCd) 9 128 PSL (Cd) (BCu) 0 (BCd) 6 ASL (Bu) 123 ASL (Bd) 282 Figure 2 Venn diagram showing numbers of genes identified as differentially expressed by microarray analysis. Summary of numbers of genes up and down regulated. The genes presented are all significantly altered in expression (P < 0.001 and multiple test correction) with >2 fold change in expression. Up regulated in AFL group relative to PFL is also denoted as Au, upregulated in ASL relative to PSL is denoted as Bu, up regulated in PSL relative to PFL is denoted as Cu, and down regulated genes are Ad, Bd and Cd respectively.