Guiry et al. BMC Genomics 2010, 11:211 http://www.biomedcentral.com/1471-2164/11/211 5× SSC, 0.1% SDS, 0.1 mg/ml BSA at 42°C for 1 h, then washed twice for 5 min with 0.1× SSC and for 30 s with 18 MΩ H2O. The slides were dried immediately by cen- trifugation (Eppendorf Multipurpose Centrifuge 5804) at 1500 rpm for 5 min at room temperature in a slide box lined with 3 MM Whatman paper. The denatured Cy dye-labelled cDNA was immediately applied to the slides. The purified, combined Cy5- and Cy3-labelled cDNAs were denatured at 95°C in a thermal cycler for 3 min. Dust was removed from the surface of the pre- hybridised arrays using an ozone-free air duster (Kenair, UK). Denatured target cDNA (80 μl) was applied to the array surface which was covered with a lifter slip (Erie Scientific, USA). Each array was placed in a pre-warmed Genetix hybridisation chamber. The chamber was sealed and incubated in an oven for 16-18 h at 50°C. Microar- ray wash solutions were warmed to 55°C. All wash steps were carried out in the dark. Each array was dipped in a 50 ml conical tube filled with wash solution 1 (2× SSC, 0.1% SDS) to dislodge the cover-slip. The arrays were washed twice for 5 min on a rotary table in fresh wash solution 1. The arrays were transferred to wash solution 2 (0.1× SSC) and washed twice for 1 min on a rotary table. Finally, the arrays were rinsed by immersion in RNase/DNase-free water to remove any residual salts, followed by brief immersion in 100% isopropanol (Sigma-Aldrich) at room temperature and brief centrifu- gation at 1500 rpm for 2 min to dry the slides. The arrays were scanned as soon as possible after washing. Microarray scanning and analysis After hybridisation, fluorescence was measured using a confocal laser scanner (ScanArray Express HT, Perkin Elmer, USA). The high-resolution images were inspected using Genepix Pro v5.1.0.16 (gonad data) or Genepix Pro v6.0.1.25 (brain data) (Molecular Devices, USA). The LIMMA package for analysis of gene expression microar- ray data was used for both experiments [23,24]. For this spots were weighted based on feature flagging in Gene- pix: weak signal and feature irregularity appeared to be the main source of discrepancy therefore features were flagged to avoid these spots. Poor quality spots were assigned a weight of 0.1 whereas remaining good quality spots were given a weight of 1.0. For the gonad analysis median local background values were subtracted directly from median feature values, dye channels were swapped as appropriate and a ‘printtiploess’ normalisation (within slide) was applied. For the brain analysis background sub- traction used the “normexp” method in LIMMA with an offset of 50, otherwise the treatment of data was the same as for the gonad experiment. LIMMA uses an empirical Bayes method for improved power in designs which only include a small number of arrays [23]. Page 16 of 18 Different design models in LIMMA allowed a global comparison of the precocious and non-precocious gonads but, in addition, the different pools (i.e. 1 to 3) could be compared to each other: this was of interest as it could highlight any variation (either technical or biolo- gical) between individual samples. For the gonad analysis we chose to treat duplicates individually and only where both spots were significant (P < 0.05) was the spot accepted. This will tend to increase the number of false negatives but decrease the number of false positives. For the brain analysis we chose to average the MA values for duplicate spots. Quantitative PCR Real-time PCR using SYBR Green-based detection was carried on the Mx3000P Real-Time PCR System (Strata- gene). PCR primers (MWG Biotech, Ger) were designed according to the recommended guidelines in the Quan- tiTect SYBR Green PCR handbook (Qiagen Ltd., West Sussex, UK) and are provided in Table 4. All primer products and PCR conditions were first optimised using standard PCR with electrophoresis on ethidium bro- mide-stained agarose gels. cDNA was generated by reverse transcription of 1 μg total RNA (whether as a pool or individual) using an anchored oligo VdT26 ran- dom primer (V9) and Stratascript RT enzyme in a total volume of 20 μl following the manufacturer’s protocol. cDNA was diluted to 100 μl before use as a template for qPCR. Initial analysis of gene expression was carried out on pooled individual cDNA samples. Technical variation was minimised by reverse transcribing all samples at the same time. The analysis was then carried out on indivi- dual cDNA samples in triplicate for each gene of inter- est. PCR data (as threshold cycle (Ct) numbers) were imported into Excel where the 2^-DeltaDeltaCt formula [36] was applied. This data was then imported into the GraphPad InStat programme (version 3.0) for statistical analysis. (Additional file 2). Additional file 1: Full version of Table 1 (Table 1s). Differentially expressed genes of the testis during precocious maturation. This version includes additional clones that were not definitively identified and includes two additional columns detailing clone library origin and sequence homology information. Additional file 2: Selection of housekeeping genes for testis and brain tissue. Further details of how qPCR analysis was carried out routinely and a discussion of how housekeeping genes were selected for testis and brain tissue. Also further details of the MCH1 and MCH2 qPCR analyses of individual brain samples are provided. Additional file 3: Full version of Table 3 (Table 3s). Differentially expressed genes of the brain during precocious maturation. This version includes additional clones that were not definitively identified and includes two additional columns detailing clone library origin and sequence homology information.