Martin et al. BMC Genomics 2010, 11:418 http://www.biomedcentral.com/1471-2164/11/418 in isopropanol and dried by centrifugation before being scanned. All 48 slides were hybridised at the same time to reduce any technical artefacts that may occur. The slides were scanned on an Axon 4200A scanner (Axon Instruments) at a resolution of 10 μm and saved as *.TIF files. Initial image analysis was by GenPix (ver- sion 5.1, Axon instruments) program. The array images were edited to ensure that the GAL (Gene Associated List) files were correctly orientated and that any abnor- mal hybridization signals were flagged as bad and the image output saved as *.gpr files. Image output files were imported into the R statistical environment [78] using the BioConductor Limma package [79] for prepro- cessing and further analysis. Individual spot quality weights were derived based on flags generated by the GenPix software and those spots flagged as bad were assigned a weighting of zero in subsequent analysis. Spot intensities were background corrected using the normexp method with an offset of 50 to avoid zero or negative intensity values [80]. Intensity dependent loess normalization was performed to balance any dye bias and print-tip effect within each array [81]. Quantile Table 7 Primers for real time PCR analysis Primer name Gene name Hepcidin TRL 5 Jun B AF542965.1 AY628755.1 CA056715.1 CAAT/EBP DW550698 COUP TF CK887094.1 Precerebellin CB511158.1 C-type lectin CB516930 SAA ELF-1a BQ037050.1 AF321836.1 Exs1F1 Exs1R1 sTRL5F1 sTRL5R1 Exs5F1 Exs5R1 Exst10F1 Exst10R1 Exs22F Exs22R Exs3F1 Exs3R1 Exs2F1 Exs2R1 Exs14F Exs14R EF1AEF EF1aER Page 17 of 20 normalization was performed to stabilize the experimen- tal variances across replicate arrays. Differential expres- sion of individual genes was assessed using linear modelling and empirical Bayes methods [79]. The linear model included the effects of bacterial infection (Aero- monas infected and PBS control), feeding regime (feed- ing and fasting) and the interaction between bacterial infection and feeding regime (equivalent to a two-way analysis of variance). Specific comparisons of treatment groups were made by extracting the appropriate con- trasts from the linear model. Comparisons were based on a robust t statistics in which the standard errors of the estimated log fold changes were moderated across genes using an empirical Bayes approach [82]. Multiple testing was accounted for by controlling the false dis- covery rate (FDR) at 5% [83]. Clones were assigned as being differentially expressed if both the FDR adjusted P values were less than 0.001 and a greater than two fold change in expression level. Details of the TRAITS/SGP microarray are available at European Bioinformatics Institute (EBI) ArrayExpress platform http://www.ebi.ac. uk/arrayexpress under accession number A-MEXP-664. GCTGTTCCTTTCTCCGAGGTG GACAGCAGTTGCAGCACAAAC GACCCCGGTGTGGCTGAA TGGCTGATTTGTTTTACGCTGT TACTGCACTGTTGGGACAGC GTTCAGTATGCCCCGAGTGT CAGCGGGTGTTAAGATCCAT GCAGCAGGAGGATCCAAGTA CATCGAAAGCCTGCAGGAGAAATC CCTACCAAGCGGACGAAGAACAGC GAGGGAACTGACAGCCAGAG TTCCCAACATTGCAAGTGAA AATCAGTTTGGCAAGCAGCAGA AAGCGATTTGAGATGTTTTAGTG CCCTGCAGGTGCTAAAGACAT CCTCGACCACTGGAACCCTGAA CAAGGATATCCGTCGTGGCA ACAGCGAAACGACCAAGAGG 55°C 55°C 55°C 55°C 55°C 55°C 55°C 55°C 55°C 163 310 171 200 165 246 374 186 327 Accession number Primer sequence Annealing temp product size PCR primers for real time PCR analysis to assay gene expression of cDNAs found to be differentially expressed by the diet and infection experiments. Amplified cDNA was monitored by Sybr green (BioRad) incorporation on an Opticon real time PCR machine.