Guiry et al. BMC Genomics 2010, 11:211 http://www.biomedcentral.com/1471-2164/11/211 Page 10 of 18 Table 3: Differentially expressed genes of the brain during precocious maturation. (Continued) GO256586 ↑ ↓ ↓ ↓ 1.2 1.2 1.7 1.9 TGFB2 VAMP-2 Vasotocin-1 Vasotocin-1 See footnotes to Table 1. In addition: 4First 539bp do not align to myelin P0-like glycoprotein (S. salar): 318/321 (99%) align to S. salar EST GE77456 2E-176 Aj318936 CA060527 Ssa.21646 DY736376 DY736376 genes showing significant differential regulation (P < 0.01) between the precocious and non-precocious brain samples, 23 also showed significant differential expres- sion between one of the biological replicates, i.e. PB1 v PB2 (12 clones) or NPB1 v NPB2 (11 clones). This is possibly a symptom of the small expression changes and subsequent limitations of the analysis; however 84% of the genes were still unique to the precocious versus non-precocious comparison. From a physiology perspec- tive it could also be argued that the brain has multiple roles to play at any one time and is therefore more affected by the environment than the testes. Individual fish may thus, depending on slight variations in condi- tions such as social status, availability of food, etc., show more variation in brain expression than in gonad expression. Interestingly all eight globin clones were included in the 12 clone overlap with the PB1 v PB2 comparison and isotocin and both vasotocin clones were included in the 11 clone overlap with the NPB1 v NPB2 comparison. Quantitative PCR on pooled brains In order to validate the findings of the microarray analy- sis on the brain tissue a number of genes were selected and further investigated using real-time quantitative PCR. These qPCR experiments were carried out on pools of five brains (either PB1 or PB2 against NPB2). The reference gene used was ubiquitin (Additional file 2). Changes suggested by microarray analysis were gen- erally confirmed by qRT-PCR, however, unidentified clone 02A07 and beta-globin, both of which appeared down-regulated on the array, were shown to be rela- tively unchanged by qPCR (Table 2). Melanin-concen- trating hormone (MCH), which was in the top 100 list, appeared to be up-regulated on the array in precocity. This gene was examined in 5 precocious individuals (PB2 pool) and in 5 non-precocious individuals (NPB2 pool). On average MCH was up-regulated approximately 1.7 fold in precocious males. Although many studies of male precocity in salmonids have reported the association of physical features of fish with life trajectories [4,5] it is only recently that a num- ber of papers have reported the use of genomic approaches to investigate genetic changes associated with these life trajectory decisions in salmonids [39-42] and other species [43,44]. The array described herein has been generated from clones which have been selected in relevant tissues for their role in processes that are likely to be involved in precocity. SSH is a com- monly used molecular technique to enrich for genes dif- ferentially expressed between two populations [21]. Here it has been used to enrich for precocity- and matura- tion-related genes in brain, brain-related and testes tis- sue. Furthermore, as the parr-smolt transformation and sexual maturation are often considered competing pro- cesses, smolting SSH libraries (for brain and related tis- sues) may also be relevant to precocity. In all 500 genes were determined to be differentially expressed between precocious and non-precocious gonad pools (P < 0.05) and 142 genes between preco- cious and non-precocious brain pools (P < 0.01). In both cases there were approximately 3 times more genes down-regulated than were up-regulated. This suggests a turning down of non-essential processes and the turning up of specific pathways. Some patterns of expression suggested the involvement of specific physiological pro- cesses. However, both the relatively small number of unambiguously identified genes and the paucity of GO terms for these meant that a thorough GO analysis was not possible. Gene expression changes in the brain were small but this is reflected by other papers on brain expression [45-47]. It is a common observation that brain expression changes are often less than 2-fold which has been noted in fish [45,48] and in other spe- cies [49,50]. Microarrays have been reported before to show smaller changes in expression than the more robust technique of qPCR and indeed we have found this ourselves in related studies [51]. Alternatively, the microarray signal represents all targets that hybridise to the probe whereas qPCR being primer-based may distin- guish between different targets and be more discriminatory. Apolipoprotein E was down-regulated in the brain whereas it had been up-regulated in the gonad (both confirmed by qPCR). It is unlikely that the specific role of ApoE is the same in both brain and testes. ApoE is involved in neural regeneration of the peripheral ner- vous system through the redistribution of cholesterol [48] therefore it is possible that it is in this role that ApoE affects brain development in the precocious male.