Guiry et al. BMC Genomics 2010, 11:211 http://www.biomedcentral.com/1471-2164/11/211 an interesting candidate. Guanine nucleotide binding pro- tein (G protein) beta polypeptide 2-like 1 (GNB2L1), also known as RACK1, plays a part in cellular signalling path- ways [37] and is known to compete with Hsp90 for bind- ing to hypoxia-inducible factor 1alpha (HIFalpha) [38]. Both alpha- and beta-globin were down-regulated in the testes in precocity and, like type 1 collagen (COL1A), they were identified more than once independently on the array. In order to add value to this data maturing adult testes sampled in July and November were also analysed: whereas November testes are likely to be almost mature (GSI ~ 2.4), July testes will be at a much earlier stage of maturation (GSI ~ 0.2). All expression levels were normalised to the succinate dehydrogenase reference gene (Table 2). AMH expression in individual precocious testes was down-regulated approximately 20- to 1070-fold in differ- ent individuals. Adult maturing testes followed a similar pattern where the more mature November adults showed high levels of down-regulation (850- to 2400- fold) and the less mature July adults showed only 3.1- to 8.6-fold down-regulation. There was more variability in the precocious levels of AMH expression than the November adults; though the number of the latter is very low (3), this might suggest that maturation is much more synchronised in the latter. AMH levels were also generally lower in the November adults than in the Table 2 Quantitative PCR validation of microarray analysis Gene Microarray qPCR Testes Expression (Fold Change) P relative to NP P relative to NP November relative to Jul AMH ApoE EF1a b-Glob Cat B b2M LPL GNBP Col 1A ApoE b-Glob IT-1 IT-2 MCH 02A07 04H011 22D02 1Clone included a microsatellite -7.2 +4.8 -1.8 -2.9 +3.2 -1.7 +2.7 -2.6 -4.0 -1.3 -1.5 -1.5 +1.2 -1.3 +1.5 +1.3 Brain Expression -58.9 +2.5 -5.3 -4.9 -2.3 -3.4 +3.7 -4.3 -16.7 -1.4 +1.3 -9.0 -9.1 +1.7 +1.1 +1.2 +3.0 qPCR -231.6 +1.2 -3.7 +3.7 1.0 -2.1 +2.9 -2.6 +5.8 Page 8 of 18 precocious fish - five of the seven precocious males had levels much higher than the three November adults. Guanine nucleotide binding protein (G protein) beta polypeptide 2-like 1 (GNB2L1) showed a similar pattern of expression to AMH, with down-regulation in preco- cious parr and in November adults compared to non- precocious parr and July adults though fold changes in expression level were small, approximately 1/10 those of AMH. Collagen (1A) and beta-globin also showed down-regulation in precocious parr but July adult levels were lower than November adults. Statistical analysis by Kruskal-Wallis and Dunn’s Multiple Comparisons tests demonstrated that expression in the precocious testes was significantly different from that in the non-preco- cious testes for all genes (P < 0.001; beta-globin beta P < 0.01) as it was also for July v November maturing adults. For AMH and GNB2L1 expression in precocious testes was similar to that of adult November testes and expression in non-precocious testes was similar to that of adult July testes (P > 0.05). For beta-globin non-pre- cocious testes were significantly different from all other testes types (P < 0.01). Microarray analysis of precocious brains Precocity effects on brain gene expression were also measured by microarray analysis. Here two pools of five precocious brains were compared directly to two pools of five non-precocious parr brains (Figure 2b). Data was analysed with LIMMA to determine which genes were differentially regulated between the pools for the two tissue types (PB v NPB). This data series has GEO accession number GSE16720. An initial list of 487 clones (P < 0.05) was reduced to a top 100 clones (on P-value) and 66 that were sequenced are presented (Additional file 3: table s2). A shortened version of this table is provided as Table 3. The fold changes in expres- sion level were small compared to changes in testes expression with a maximum of 1.7 fold up-regulation for an unknown EST and 1.9 fold down-regulation for vasotocin-1. Approximately 50% of the top 66 clones were derived from brain-related libraries. Alpha-globin was identified five times on the array and beta-globin three times: both genes showed consistent down-regula- tion of 1.4 fold and 1.5 fold respectively. Other genes that were identified more than once included beta-tubu- lin (2×), elongation factor 1 alpha (3×), apolipoprotein E (2×), suppressor of G2 allele of SKP1 (2×) and vasoto- cin-1 (2×); the only inconsistency was that one copy of G2 allele of SKP1 was up-regulated 1.4× and the other copy was down-regulated 1.3×. Several of the clones represented definite ESTs which were highly homolo- gous to other ESTs in the NCBI and TIGR databases but for which identities were ambiguous. Again between pool variation was examined in LIMMA. Of the 142