Martin et al. BMC Genomics 2010, 11:418 http://www.biomedcentral.com/1471-2164/11/418 and has impacts on protein turnover and cholesterol metabolism. The reallocation of and control of amino acids from normal physiological functions to production of APR proteins is probably the greatest effect we have observed. The starved fish have generally decreased transcription of immune genes but following infection they attempt to recover this, although they have less cir- culating plasma proteins and hence may have more dif- ficulty in achieving this and potentially expend considerably more protein and energy reserves attempt- ing to deal with the pathogen. This experiment has used an extreme model (starvation and infection) and only examines the mRNA responses for those genes present on the microarray. Future experiments could incorpo- rate additional physiological and immunological para- meters. The results presented here may help predict how fish will respond to infection or vaccination on specific diets and the importance of high protein feeds during times of a potential immune challenge. Methods Fish maintenance and challenge Juvenile mixed sex Atlantic salmon were maintained in 250 L freshwater tanks at the University of Aberdeen, UK, fish facilities. Water was held at a constant 12°C, pH 7.60 (± 0.05) and 90% (± 1%) of oxygen saturation. The fish were individually marked on the ventral surface by alcian blue dye, which enabled recognition of indivi- duals. Initially the fish were fed ad libitum (Nutreco feed). At the beginning of the experiment fish were separated randomly into two tanks, in one tank fish were fed ad libitum as before and in the other tank they were not fed for 4 weeks. The initial and final weight and length were recorded and were used to calculate condition factor (K, 100 * BW/FL-3) where BW is body weight in g and FL is fork length in mm. Specific growth rate (SGR) was calculated from the equation: SGR = ((LnBW2 - LnBW1)/t) × 100 where BW2 is the final weight, BW1 is the initial weight and t is time between W1 and W2 in days. Following the 4 week trial fish were anaesthetised with bezocaine (Sigma 20 mg l- 1) and injected with 100 μl of a genetically attenuated (aro A-) strain of Aeromonas salmonicida (Brivax II[77]) (109 CFU ml-1) in PBS or 100 μl of PBS as control. Fish were returned to separate tanks, either infected or con- trol. The fish were then sacrificed 24 h post challenge and liver tissue was removed immediately and stored in RNAlater (Ambion) at 4°C for 24 h then placed at -20°C until RNA extraction. A total of 96 fish were used in the trial, split into four groups of 24, denoted as AFL (Aeromonas infected/fed), ASL (Aeromonas infected/ starved), PFL (PBS control/fed) and PSL (PBS control/ starved). Page 16 of 20 RNA isolation Liver tissue (100 mg) stored in RNAlater was blotted on tissue paper to remove any excess liquid. The tissue was then homogenised in RNA STAT60 (AMS Biotechnol- ogy) according to the manufacturer’s instructions for purification of total RNA. After precipitation, RNA was resuspended in sterile RNase free water (Sigma). Further RNA purification was performed using RNeasy cleanup columns (Qiagen) according to the manufacturer’s pro- tocol. RNA was eluted from these columns in water and the concentration adjusted to 2.5 μg μL-1. The integrity of the RNA was determined by electrophoresis (Agilent Bioanalyser 2100) and concentration measured by spec- trometry (ND-1000, NanoDrop Technologies). RNA was stored at -80°C until required. Microarray hybridisation and analysis For microarray analysis 6 pools of RNA were made, each pool containing RNA from 4 different fish chosen randomly from each group. Concentration and quality was rechecked as described above. RNA was reverse transcribed using a FAIRPLAY II cDNA post labelling kit (Stratagene) according to the manufacturer’s instruc- tions and labelled separately with both Cy3 or Cy5 (GE HealthCare; PA23001, PA25001) to allow for a swap protocol. All reverse transcriptions were carried out at the same time on 96 well plates to reduce technical var- iation. Briefly 20 μg total RNA was reverse transcribed after being primed with oligo dT. Following reverse transcription the RNA template was hydrolysed using 1 M NaOH for 15 min and then neutralised with 1 M HCl. The cDNA was ethanol precipitated overnight. The cDNA pellets were washed in 80% ethanol and air dried before being resuspended in 5 μl 2× coupling buffer. Once the cDNA had fully dissolved (after at least 30 min) 5 μl Cy dye was added to each tube and incubated in the dark for 30 min. (Pre-aliquotted Cy3 and Cy5 dyes were resuspended in 45 μl DMSO prior to being added to the coupling buffer.) To remove unincorpo- rated dye, the labelled cDNA (total volume 10 μl) was passed through a DyeEx 2.0 spin column (Qiagen). Dye incorporation was checked by spectrophotometry (ND- 1000) and by electrophoresis of labelled cDNA on a mini-gel and visualisation by microarray scanner (Perkin Elmer ScanArray 5000XL). For hybridisation, 20 μl of labelled sample (10 μl Cy3 and 10 μl Cy5) was added to 85 μl hybridisation buffer (Ambion), 10 μl poly(A) potassium salt (Sigma; 10 mg ml-1) and 5 μl ultrapure BSA (Ambion;10 mg ml-1). Hybridizations were performed on a Gene TAC Hyb Station (Genomic solutions) for 16 h at 42°C. Following the hybridisations slides were washed with 1× SSC 10 min at 60°C, 1× SSC +0.2% SDS 10 min at 60°C, 0.1 × SSC + 0.2% SDS 10 min at 42°C. Slides were then rinsed