Guiry et al. BMC Genomics 2010, 11:211 http://www.biomedcentral.com/1471-2164/11/211 considered whether some of the identified genes differed in expression between precocious males and returning males. Results and Discussion Microarray analysis of precocious testes Non-precocious parr were significantly longer and hea- vier than precocious parr (Figure 1). The difference in weight (using an unpaired t-test) and length (using a Mann Whitney test) in the two populations were both shown to be significant (P < 0.0001). It is generally accepted that males which grow at a faster rate are more likely to become precocious than slower growing male parr [19]. However, once this life history choice has been made (certainly by January), sexual maturation proceeds at the expense of somatic growth. Total RNA, isolated from precocious and non-preco- cious parr testes was shown to be intact by both North- ern blot and analysis using the Agilent 2100 Bioanalyser. All samples used subsequently had RIN values between 9 and 10 [20]. Suppression Subtractive Hybridisation (SSH) was used to enrich for genes differentially expressed during the process of precocious maturation [21]. Two pairs of libraries related directly to this study of precocity (testes and brain) while other SSH libraries related to different tissues (e.g. hypothalamus and pitui- tary) or different aspects of sexual maturation (e.g. adult maturing ovary). Furthermore, there was a selection of testes full-length (not SSH) and undifferentiated gonad Page 3 of 18 (SSH) clones from other sources both salmon [22] and trout (McMeel and Guiguen, unpublished). Three pools each of four individual precocious testes were compared by microarray analysis to three pools of ten pooled non-precocious testes (Figure 2a). Although not allowing a direct comparison of individual fish this loop design provided scope to look at individual pools yet maintain a compact design with dye balance and a minimum number of hybridisations [23,24]. This data series has GEO accession number GSE16721. A list of significant differentially expressed clones (P < 0.05 and using the Benjamini and Hochberg multiple test correc- tion) was compiled. This list of 500 clones was cut down to approximately 65 for sequencing based primar- ily on fold changes in expression (Additional file 1: table s1). A shortened version of this table is provided as Table 1. Most clones (75%) showed homology to known database sequences, however 17% were found to have homology to unidentified ESTs and 8% ‘failed’ sequen- cing. ‘Failed’ sequences encompassed short sequences (e.g. too short to identify by homology searches), poor sequences (e.g. poly Ts and mixed clones), and no sequence (template problem). Between pool variation was also examined in LIMMA. For the P pools 484 of the 500 top clones were identified in all three pools and 499 in at least two of the pools. For the NP pools 499 of the top 500 clones were identified in all three pools and all 500 in at least two of the pools. This indicated that no individual sample was excessively Figure 1 Fish sample populations for precocity and non-precocity. Precocious fish (empty circles) and non-precocious fish (full squares) were weighed and measured. Fish (25 of both precocious and non-precocious) were sampled on 15 Jan 2003. The difference in weight (using an unpaired t-test) and length (using a Mann Whitney test) in the two populations were both shown to be extremely significant (P < 0.0001).