Martin et al. BMC Genomics 2010, 11:418 http://www.biomedcentral.com/1471-2164/11/418 The raw hybridisations data have been deposited at ArrayExpress under accession number (E-MEXP-2496). All experimental procedures for array construction, hybridisation and analysis comply with MIAME guide- lines [84]. Analysis of gene ontology Enrichment for specific gene ontology (GO) categories was performed on all those cDNA features that had associated GO identifiers using the GOEAST program [85]. Fisher’s exact test was used within the GOEAST program to determine if GO identifiers occurred more often in a group than would appear by chance. For GO analysis only biological process GO identifiers were con- sidered that occurred more than 4 times. Real time PCR All RNA samples for real time PCR were taken from the same pool of material as used for microarray analysis. RNA (2 μg) was denatured (65°C, 10 min) in the pre- sence of 1 μl oligo dT17 primer (500 ng μl-1), left at room temperature for 5 min to allow annealing, then kept on ice. cDNA was synthesised using 15 U Bioscript reverse transcriptase (Bioline, UK) in the presence of dNTPs (final concentration 200 μM each), at 42°C for 1 h in a final volume of 20 μl. The cDNA was diluted to 100 μl and 3 μl used as template for PCR using primers designed against the Atlantic salmon genes of interest (Table 7). cDNA amplification using ready-prepared 2× SYBR Green PCR master mix (Biorad) was performed in 25 μL volumes on a white BioRad 96 well PCR plate covered with transparent film, and an Opticon qPCR machine was used for monitoring cDNA amplification. A negative control (no template) reaction was also per- formed for each primer pair. Efficiency of amplification was determined for each primer pair using 10 fold dilu- tions (1, 10, 100 & 1000 fold dilutions). Elongation factor 1a was used for normalization of expression, previously validated for real time PCR [86]. PCR conditions for all gene assays were 95°C for 5 min followed by 94°C for 15 s, 57°C for 15 s, 72°C for 20 s for a total of 35 cycles. The fluorescence signal output was measured and recorded at 78°C during each cycle for all wells. Melting curves (1°C steps between 75°C - 95°C) ensured that only a single product had been amplified in each reaction. A sample from the serial dilution was separated on an ethidium bromide stained agarose gel, to confirm that a single band of correct size was amplified. To determine the relative expression level of candidate genes the method described by Paffle [87] was employed using elongation factor 1a as house keeping gene. The efficiency of the PCR reaction for each primer set was measured on the same plate as the experimental sam- ples. The efficiency was calculated as E = 10(-1/s) where s is the slope generated from the serial dilutions, when Log dilution is plotted against ΔCT (threshold cycle Page 18 of 20 number). For all real time PCRs six replicates were performed and statistical analysis performed by t-test. Additional material Additional file 1: Table S1. Full list of genes altered significantly in following infection and starvation. Additional file 2: Table S2. GO Biological processes enriched following starvation and infection. Additional file 3: Table S3. Genes altered significantly in liver by infection. Additional file 4: Table S4. Full list of genes responding differently to infection between fed and starved fish. Acknowledgements This work was supported by BBSRC grant EGA17675 (Salmon TRAITS). We thank James Bron and John Taggart for microarray annotation and Janet Davidson for critically reading the manuscript. Authors’ contributions SAM performed microarray experiments, analysed the data, carried out the real time PCR and wrote the manuscript. AD performed the microarray data analysis. DFH and CJS were involved in the experimental design and drafting of the manuscript. All authors read and approved the final manuscript. Received: 23 December 2009 Accepted: 5 July 2010 Published: 5 July 2010 References 1. Beutler B: Innate immunity: an overview. Molecular Immunology 2004, 40(12):845-859. 2. Magnadottir B: Innate immunity of fish (overview). Fish & Shellfish Immunology 2006, 20(2):137-151. 3. Bird S, Zou J, Secombes CJ: Advances in fish cytokine biology give clues to the evolution of a complex network. Current Pharmaceutical Design 2006, 12(24):3051-3069. 4. 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