BMC Genomics 2009, 10:490 http://www.biomedcentral.com/1471-2164/10/490 TFihgisufriegu3re clarifies which of the methods was optimal for finding human-carp orthologs This figure clarifies which of the methods was optimal for finding human-carp orthologs. CSRBH performed best with 52% of contiguous sequences successfully assigned an ortholog as against 49% and 41% for SRBH and RBH respectively. method, reduces the high percentage of angiogenic genes from 25% down to 9. This is reduced further if you only take genes with 3 and/or 4 methods. But given the full list, a researcher can decide based on the presence of func- tional domains or personal knowledge which ones are of interest to investigate with full knowledge that the human functional homolog was only loosely associated with the biological process of interest. Re-analysis of gene expression data Three established methods of measuring gene expression used in biological research are microarrays, cDNA and Serial Analysis of Gene Expression (SAGE) libraries. To investigate numerous pathologies many institutes around the world have generated, analysed and deposited large amounts of expression data into public repositories [57]. Examples of such databases are the Gene Expression Omnibus (GEO), Cancer Genome Anatomy Project (CGAP), National Center for Biotechnology Information (NCBI) and European Bioinformatics Institute (EBI) arrayExpress. It has been shown that data mining of rele- vant data sets can successfully lead to the identification of biologically interesting targets [15,16,58-67]. Cold response differentially expressed genes Carp is an ectotherm with a body temperature similar to the water it is immersed in and can adapt to a range of temperatures (eurythermal). A recent study investigated physiological adaptation of carp exposed to gradually lower temperatures [26]. Microarray analysis measured the change in gene expression, in several tissues, at several cooling stages from 30 to 10°C vs. a control temperature of 30°C. Overall, 252 transcriptional regulatory, RNA splicing and translation control genes were found to be up-regulated in all tissues. Interestingly, some tissue-spe- cific affects were also seen, e.g. in the glycolytic pathway of brain and lipid metabolism of liver. This data set proved appropriate for re-analysis to predict cold-induced angiogenic genes in skeletal and heart muscle tissues. Persistently up-regulated genes Two strategies were applied to the data. The first utilised the data without intervention for heart and muscle tissues, employing a threshold score > = 2. The total number of up-regulated cDNAs for heart was 748, 270 non-redun- dant genes, and for muscle 1230 cDNAs, 475 genes. Any cDNAs with significant contradicting expression were removed. For both heart and muscle combined there were 589 non-redundant genes, 143 of which were predicted angiogenic genes (see table 3 and additional file 12; col- umns c and d for both heart and muscle sheets). Initial cooling, fold ratio subtraction and microarray re- analysis It is thought angiogenesis is switched on in the initial stages of cooling, before the animal has become acclima- tised to a particular temperature. This has been shown in rats subjected to cooling to 4°C which led to a 2.7 fold increase in VEGF expression for 1 to 4 hours and returned to the basal level at 24 hours [8,9]. Therefore, the micro- array data was analysed between the first and last time point at each target temperature. Page 8 of 17 (page number not for citation purposes)