Martin et al. BMC Genomics 2010, 11:418 http://www.biomedcentral.com/1471-2164/11/418 also down regulated following infection, although again, only significantly so for the ASL group. Starvation and infection interaction A key feature of this study was to determine if the feed- ing regime prior to infection had any effect on the change in the liver transcriptome following infection. The difference in the response between the ASL and AFL treatment groups was examined by extracting this specific contrast (ASL vs AFL) from the linear model. This resulted in the identification of 474 genes (198 at greater than 2 fold) that responded differently to infec- tion depending on prior feeding. From this analysis 103 and 53 genes showed a greater expression in AFL and ASL respectively. The results from the linear model gave 4 possible groups (Table 4, Additional file 4 table S4); 1. increased more in AFL than ASL (51 genes), 2. increased more in ASL than AFL (26 genes) 3. decreased more in ASL than in AFL (51 genes), and 4. decreased more in AFL than ASL (27 genes). For GO analysis to examine the interaction between infection and feeding regime, both those genes that responded at a greater or decreased level were com- bined. Fourty eight GO biological processes were signifi- cantly altered ie. had a different magnitude in infected fish depending on if they were fed or starved before the infection (Table 5) and included genes involved in immune function, protein metabolism, lipid metabolism and cellular homeostasis. Genes encoding proteins that are secreted into plasma were affected to the greatest extent between ASL and AFL. Complement components H, B and C1 were more induced in AFL whereas complement C7 showed greater up regulation in ASL. A number of major acute phase protein genes, serum amyloid A (SAA), serotransferrin and b 2 microglobulin (b 2 M) were all highly up regu- lated to a significantly greater magnitude than in the AFL group, suggesting a greater increase in production of these proteins. Hepcidin also had a 3.8 fold greater increase in ASL than AFL showing again that the ASL group responded differently to the AFL group. It is also of note that both SAA, hepcidin and serotransferrin were decreased in expression in the starved group (PSL). This additional increase in the ASL group compared to AFL may be a compensatory increase due to the decreased expression of these genes in the starved fish. Cell surface receptors including Toll like receptor 5 was up regulated to a greater extent in ASL than AFL, whereas there was a less clear picture with the C type lectins. Thus, maltose binding lectin was down regulted in ASL to a greater degree than in AFL whereas a C type lectin receptor A was more highly up regulated in ASL than AFL. Page 9 of 20 Genes in the steroid biosynthesis/cholesterol pathway which includes IIP isomerase, steroidogenic acute regu- latory protein and lathersterol oxidase were up regulated to a greater extent in the AFL. Expression of genes related to amino acid metabolism were reduced in both ASL and AFL, but a greater reduction was seen in serine pyruvate amino transferase and tyrosine amino transfer- ase where there was a significantly greater reduction in ASL fish. Confirmation of expression by real time PCR Real time PCR analysis was performed on a number of genes for confirmation of microarray data (Table 6). All the genes were those that showed a significant increase in expression following the infection challenges, and represented genes related to different aspects of the immune response. These were SAA, Toll like receptor 5, hepcidin, JunB, CAAT, COUP, C type lectin and precerebellin. The qPCR expression was normalized using elongation factor 1 a as this was not found to be modulated in expression by microarray analysis. For all genes the expression pat- tern showed the same direction of response between microarray and real time PCR analysis, however the magnitude of the expression differences varied between the microarray and qPCR. In general greater differences were observed from real time expression data than from microarray data. For example, a 47 fold increase was found for hepcidin AFL in samples by microarray analy- sis but ~100 fold difference was found when these sam- ples were examined by qPCR. Similar differences were also found for SAA. The reason for the difference is likely to be the different platforms being used and different protocols for cDNA synthesis and equipment. Discussion Both infection and food withdrawal have major conse- quences to whole animal physiology with alterations occurring in many cellular processes across numerous tissues. Central to metabolism is the liver, which carries out many processes related to energy metabolism, synth- esis and secretion of serum proteins and immune responses. During infection the liver has a number of essential roles, in particular the production of acute phase response proteins (APR) [33] as well as specialized liver specific macrophages, the Kupffler cells [34]. Both starvation and infection have been examined in fish, but rarely has the interaction of these two processes been investigated. A considerable amount of literature is available on the transcriptional response to starvation in different fish species, however care needs to be taken when interpreting the results as many factors including, prior feeding [26], stress [35], energy reserves [36] and