BMC Genomics 2009, 10:490 http://www.biomedcentral.com/1471-2164/10/490 replicates and as such, there were no real biological repli- cates. Therefore, the comparison between the methods might be more informative. In the results genes that are predicted by 1 method and not contradicted by the others are used: this is equivalent to lowering the multiple test correction threshold in one method when results agree with the other. Total cold induced genes 2035 cDNA sequences were unambiguously up-regulated for all three methods, representing 698 genes and 171 of these were identified as angiogenesis-related genes (table 3 and additional file 12). Summary of analyses The combined results of this analysis are summarised in figure 4 and are as follows: From the 16,650 contigs searched, 8,726 were successfully assigned an ortholog, which amounted to 3,762 non-redundant gene orthologs. 908 of the orthologs were predicted to be angiogenesis genes and 698 of the predicted orthologs were found to be induced by cold temperature. Finally, cross-referencing these genes found 171 cold induced angiogenic genes (additional file 13). Biologically relevant genes The aim of this study was to develop a bioinformatic method of predicting genes of biological interest in a non- model species. Although many putative angiogenesis genes were found, some key angiogenic genes were not present on the carp array. For instance, vascular endothe- lial growth factor (VEGF) is a major activator of angiogen- esis [69] and it would have been interesting to see the VFeignunrdeia4gram summary Venn diagram summary. The Venn diagram summarises the results from the different searches and analyses. 171 cold induced angiogenesis genes were predicted in this study. effect of cold exposure on this gene. Nevertheless, table 4 displays 12 out of the 171 angiogenesis-related genes up- regulated by low temperature. On investigation of these genes (highlighted on table 4 and below), there is encour- aging evidence that these methods were successful. Mitogen-activated protein kinase 1 (MAPK1) MAPK1 is part of the MAPK-signalling pathway, which is the interface to a myriad of cellular processes like differen- tiation, proliferation and development [70-72]. It is also activated by two upstream kinases RAF and MEK, which are themselves activated by growth factor receptors [72]. Upon activation, MAPK1 is phosphorylated and translo- cates to the nucleus where it phosphorylates targets such as c-Fos, c-Jun, c-Myc and Tal-1 and initiates transcription of many genes [72]. During angiogenesis MAPK1 is acti- vated in ECs and induces EC proliferation, critical to the establishment of new vessels [73], abrogates apoptosis and promotes the transcription of VEGF, itself a key acti- vator of angiogenesis [74]. It is possible this is done via the hypoxia-inducible factor 1 alpha (HIF1A) gene as MAPK1 can activate HIF1A and this transcription factor is known to mediate VEGF expression [71,74]. The involvement of HIF1A in cold-induced angiogenesis fits well as reduced oxygen diffusion and increased blood viscosity can lead to tissue hypoxia. Interestingly, the MAPK-pathway regulates expression of MMP9 [75,76] which also was predicted as a cold-induced angiogenic gene in this study. This finding is supported by the fact that inhibition of MEK, an upstream activator of MAPK1, curtails the expression of MMP9 [75] and that the MAPK-pathway induces transcription of MMP9 via the upstream promoter site AP-1 [77]. Matrix metallopeptidase 9 (MMP9) MMP9 is a member of a family of zinc containing endopeptidases (type IV collagenase) and plays a major role in angiogenesis [78,79]. MMP9 is synthesised as an inactive zymogen (pro-MMP9) that is activated by prote- olysis [80]. During angiogenesis, MMP9 is secreted by endothelial cells to degrade the extracellular matrix, thus allowing ECs to migrate and form new blood vessels [81]. Coupled to this are the facts that MMP9 inhibition signif- icantly decreased cerebral EC migration and proliferation [82] and that a positive correlation with micro vessel den- sity and MMP9 expression exists [83]. MMP9 is also known to release VEGF from extracellular matrix (ECM) stores [84,85]. Ras homolog gene family, member A (RHOA) Another cold-induced angiogenic gene that was predicted in this study was RHOA, which is also known to induce MMP9 expression [86]. RHOA is a member of the RHO- family GTPases which are part of the RAS superfamily Page 10 of 17 (page number not for citation purposes)