Guiry et al. BMC Genomics 2010, 11:211 http://www.biomedcentral.com/1471-2164/11/211 (transfer in the first week of May). There may therefore be some changes in brain precocity expression which are confounded by brain smoltification expression. In a separate experiment (White et al., personal communica- tion), the same genes were followed in a brain smolt time-course from January to May. From this it is clear that most of these genes do not change significantly over the period of smoltification and therefore it is unlikely that down-regulation in precocity is really up- regulation in smoltification. It is however interesting that in the Aubin-Horth study several genes, including beta-actin, cathepsin D, an elongation factor, beta tubu- lin and both vasotocin and isotocin, showed relative up-regulation in ‘early migratory’ fish that also showed up-regulation in our immature parr. The fish used in this study are from a hatchery-reared stock that has been developed from a small number of originator males and females and should therefore be of relatively homogeneous genotype. We do not necessarily expect that hatchery and wild salmon will behave in the same way. It has been shown that the rearing environ- ment of Atlantic salmon has an effect on gene expres- sion and additionally that this effect interacts with mating tactic [39]. In a study that used a cichlid micro- array to compare wild and laboratory reared salmon from the same source it was clear that some differences in expression between immature male brains and preco- cious brains were dependent on the rearing environment [39]. This in itself suggests that the sneaker tactic/phe- notype does not develop by one unique route and there- fore that gene expression profiles may differ from one individual to another due to environment. Also it must always be remembered that for many regulators such as isotocin and vasotocin, because of storage, secretion and blood clearance considerations, transcript levels in the brain and gonad may not directly correlate with protein levels in the plasma, and that plasma levels do not necessarily reflect activation of the system as the hor- mone receptor in the effector tissue may be down-regu- lated to prevent effects [57]. As noted above, isotocin and vasotocin blood plasma levels may be high leading to activation of a pathway or process but transcript levels could be low because either the hormone was released from stores (not requiring de novo transcrip- tion) or a feedback mechanism was down-regulating transcription. It is a complicated issue where the specific biology is known to vary between different fish species and even within species of the family Salmonidae Conclusions A custom-built salmonid cDNA microarray has been used to compare expression changes in the brain and gonad during the process of precocious sexual matura- tion in Atlantic salmon. Transcriptional regulation in Page 12 of 18 the brain was minor compared to the testes. Microarray analysis suggested a general down-regulation of house- keeping functions and more specific up-regulation of a number of genes. Distinct expression profiles for both brain and gonad will be determined by maturation stage (recrudescent, mature, regressed) and environmental factors. Anti-Mullerian hormone plays a major role in the development of the maturing gonad as too does lipid mobilisation through the apolipoproteins and lipo- protein lipase. Methods Sample collection The Marine Institute in Newport, Co.Mayo, provided Atlantic salmon samples. The Marine Institute, Furnace, Newport, Co. Mayo is a registered premises under the Cruelty to Animals Act 1876, as amended by EU Regu- lations 2002 & 2005 and is declared to be a registered place for the performance of experiments. The Bur- rishoole ranch stock used in this study were originally derived from Burrishoole wild stock and have been line bred since the early 1970’s as part of an on-going experimental ranching programme. At sampling, fish were transferred to an anaesthetic/ sedative solution (ethyl 4-aminobenzoate; 0.1 g/l; Sigma- Aldrich) and killed by spinal section. Fifty precocious male parr and fifty non-precocious male parr (sex deter- mined on dissection) were sampled in January 2003 to provide brain and gonad tissue for SSH library construc- tion. Length and weight measurements were taken. Additional fish for microarray and qRT-PCR analysis were harvested in January 2004. Adult salmon were sampled from upstream traps located on the Burrishoole river system on 31 July 2003 and 25 November 2003 - these were tagged therefore are hatchery-reared fish (not wild). The gonadosomatic index (GSI) - the ratio of the weight of the gonad to that of the whole fish (× 100) - for the July fish was 0.19 ± 0.04 standard deviations (SD) and for the November fish was 2.44 ± 0.45 SD. All tissues were removed to RNAlater or snap frozen in liquid nitrogen. Samples were stored at -80°C until RNA extraction. Extraction and analysis of RNA All samples were lysed and homogenized in TRIzol reagent (Invitrogen Life Technologies, Paisley, UK) [63]. Qiagen RNeasy midi columns and wash buffers (Qiagen Ltd., West Sussex, UK) were used to purify the RNA. All samples were DNase treated on-column using the Qiagen DNA-free DNase Kit (Qiagen Ltd., West Sussex, UK). The concentrations of the RNA samples were determined by UV spectrophotometry (Shimadzu UV- 1601 or Eppendorf BioPhotometer) and checked for quality using the Agilent Bioanalyser-derived RNA