Guiry et al. BMC Genomics 2010, 11:211 http://www.biomedcentral.com/1471-2164/11/211 integrity number (RIN) (Agilent Technologies Ireland Ltd., Dublin, Ireland) and by denaturing formaldehyde gel electrophoresis followed by northern blotting and subsequent analysis with a radiolabelled beta-actin probe. Construction of Suppression Subtractive Hybridisation (SSH) cDNA libraries Tissues used for the SSH libraries were brain, pituitary, hypothalamus, head kidney, spleen, intestine, gill, testes and ovary. Libraries related to this paper were from brain, pituitary, hypothalamus and testes tissues com- paring male parr to precocious parr. Other SSH libraries of relevance compared the ovary of maturing adult females (July) and near maturity females (November). Other SSH libraries to be described elsewhere relating to the parr-smolt transformation were of brain (Feb v sea cage), hypothalamus (Feb v Apr), pituitary (Feb v Apr, Apr v sea cage), gill (Apr v May), and intestine, head kidney and spleen (Feb v May). Exact dates were 13 Feb, 4 Apr, 2 May and 27 May (sea cage) 2003. An additional library pair related to the post-blastula transi- tion of the salmon embryo. For testes tissue pools of precocious parr and non-pre- cocious parr total RNA were each prepared from 10 individual fish. For brain tissue pools of precocious parr and non-precocious parr total RNA were each prepared from 4 individual fish. For hypothalamus tissue pools of precocious parr were prepared from 3 individual fish and non-precocious parr from 3 pools each of 3 fish (29 Jan). For pituitary tissue pools of precocious parr and non-precocious parr total RNA were each prepared from 10 individual fish (29 Jan and 13 Feb respectively). The ovarian library from maturing adults and the brain library from smolting parr were made directly from mRNA isolated from total RNA using the Oligotex mRNA Isolation Kit (Qiagen, UK). All other libraries were made from SMART cDNA generated by reverse transcribing 1 μg of total RNA as detailed in the SMART cDNA Synthesis Kit (BD Biosciences, Oxford, UK). The PCR-Select cDNA Subtraction Kit (BD Bios- ciences, Oxford, UK) was subsequently used to prepare both forward and reverse subtracted libraries from each tissue. Subtracted target cDNA products were ligated into the pCR2.1 TA Cloning Vector (Invitrogen Life Technologies, Paisley, UK) using T4 DNA ligase (New England Biolabs) according to the manufacturer’s instructions. Ligations were transformed into Ultra- comp™ One-Shot Top 10F’ chemically competent E. coli cells (Invitrogen Life Technologies, Paisley, UK). Recom- binant white clones from each library (four 96-well plates for each library) were randomly selected from the plates, cultured in LB broth in 96-well plates and frozen in glycerol for archival storage at -80°C. Clones were Page 13 of 18 PCR amplified using 2.5 units of Biotaq (Bioline, UK) in a total volume of 75 μl detergent-less buffer (30 mM Tricine, pH 8.0 containing 50 mM KCl, 2 mM MgCl2, 0.2 mM dNTPs and 0.2 μM of each SSH nested primer). PCR products were checked for single products on ethi- dium bromide stained agarose gels and spotted directly. Microarray design and construction The cDNA microarray contained clones from a number of Suppression Subtractive Hybridisation (SSH) cDNA libraries [21] constructed from different salmon tissues at different developmental stages related to sexual maturation, smoltification and embryo development. Of the 5376 cDNA clones, 4128 were newly generated sub- tracted cDNA clones and the remaining 1248 clones were other SSH or full-length clones from related sal- monid projects. All SSH clones average 600-900 bp in length. Several candidate genes and controls were also included. At the printing stage DNA sequences were only known for those clones that were used to check library quality/redundancy and for those clones that ori- ginated from related salmonid projects. Otherwise clones were only sequenced after microarray analysis had identified a differentially expressed gene. The top 100 clones approximately from each of the two microar- ray analyses detailed here, and the same for additional analyses to be detailed elsewhere, were sequenced (AGOWA, Berlin, Germany). Sequences were edited to remove vector and adaptor sequences, and analysed by BlastN and BlastX algorithms against the NCBI data- bases of non-redundant nucleic acids and the protein and EST accession databases http://www.ncbi.nlm.nih. gov/BLAST/[64]. BlastN was also carried out against the TIGR database http://compbio.dfci.harvard.edu/tgi/ cgi-bin/tgi/Blast/index.cgi. The printing onto amino-silane coated glass slides was carried out at the Liverpool Microarray Facility (University of Liverpool, UK) and consisted of dupli- cate arrays with sub-arrays in a 4 × 4 format with 336 spots per sub-array. The composition of the microarray by tissue and developmental stage is given in Figure 4. The array platform has been submitted to Gene Expression Omnibus with accession numbers GPL8731 (10752 features) and GPL8704 (5376 fea- tures): the duplicate features in the gonad analysis were not averaged where they were for the brain analy- sis, hence the two platforms. Hybridisation design Non-precocious parr testes were small and collected in pools of ten. Precocious testes were more substantial and were RNA extracted individually. To compare testes sam- ples precocious parr testes RNAs were pooled in three pools of four individuals and compared in an optimised