Guiry et al. BMC Genomics 2010, 11:211 http://www.biomedcentral.com/1471-2164/11/211 Page 14 of 18 Figure 4 Composition of the salmon maturation and smoltification cDNA microarray. The array is described on the basis of the clone origin: (a) by tissue type, (b) by developmental stage and project source, and (c) by tissue library related to sexual maturation. Most clones originate from salmon SSH libraries related to sexual maturation and parr-smolt transformation, however 576 clones are of rainbow trout origin and relate to confinement stress in liver, brain and pituitary (European Union-funded STRESSGENES project, http://www.irisa.fr/stressgenes/), undifferentiated gonad and stimulated head kidney leucocytes (both included under ‘Other’). A number of clones are also from unselected salmon full-length cDNA libraries of testis, ovary, spleen and liver (European Union-funded SALGENE project). Further details are given in Materials and Methods. loop design http://www.math.rug.nl/~ernst/book/smida. html[65] to three pools of ten non-precocious parr (Figure 2a). Nine microarray hybridisations were carried out in total as part of this design. To compare brain samples total RNA from 10 individual brains (dissected for hypothalamus and pituitary) of precocious fish and 10 brains of non-precocious fish were pooled to give two pools each of five precocious (PA and PB) and non- precocious (NPA and NPB) brains. Each pool of preco- cious brains was compared in a dye swap experiment with each pool of non-precocious brains (Figure 2b). An indirect labelling method was used to attach Cy5 and Cy3 dyes to the cDNA. All target cDNAs were synthesised in one round. 10 μg of both tester and refer- ence total RNA was used per hybridisation. cDNA was generated by reverse transcription using an anchored oligo VdT26, random primer (V9) and Stratascript RT enzyme. Amino-allyl (aa) dUTP/dNTPs produced from a 20× 1:1 stock of dUTP/dTTP were incorporated into the cDNA. This 1:1 ratio of dUTP/dTTP proved to be an effective ratio for the testes tissue. Contaminating enzymes and buffers were removed by purification with the Nucleospin PCR Clean-up Kit (Macherey-Nagel, Ger). Eluted cDNA was concentrated using a Vacufuge Concentrator 5301 at 42°C (Eppendorf, Ger). The result- ing cDNA pellets were resuspended in 5 μl of sodium bicarbonate (0.1 M) (Sigma Aldrich). Cy dyes were resuspended in the same buffer and coupling to the Cy dye ester took place in the dark for 2 h at room tem- perature. The removal of unincorporated dye was per- formed using the Illustra CyScribe GFX Purification Kit (GE Healthcare). After coupling the amino-allyl (aa) dUTP-labelled cDNA to the Cy dyes (Amersham Phar- macia Biotech, UK), the labelled cDNA was again puri- fied to remove any un-coupled Cy dye (Macherey Nagel, Ger). Slides were pre-hybridised in 50% formamide,