BMC Genomics 2009, 10:490 http://www.biomedcentral.com/1471-2164/10/490 Table 3: A numerical breakdown of cDNAs differentially expressed due to cold temperature Heart tissue Muscle tissue Combined results Persistent cDNAs Persistent genes Persistent angiogenic genes Ratio cDNAs Ratio genes Ratio angiogenic genes Limma cDNAs Limma genes Limma angiogenic genes Combined cDNAs Combined genes Combined angiogenic genes 748 270 73 3 1 1 7 4 3 755 272 74 1230 475 102 82 29 7 577 195 62 1671 597 139 1603 589 143 83 30 8 581 196 62 2035 698 171 This table conveys the numerical breakdown of results for the differential cDNA expression due to cold temperatures on the carp array. 171 were the total number of putative cold induced angiogenic genes. First, ratios were calculated between each of the cooling time points vs. a control group (see methods). Analysing heart and muscle separately and combining the results gave 83 unambiguous cDNAs with a ratio subtraction > = 2 and this represented 30 non-redundant genes. 8 of these genes were predicted angiogenic, see table 3 and addi- tional file 12, column e for both heart and muscle sheets. To further discover genes switched on at initial cooling, microarray analysis using Limma from the BioConductor R package [68] was used to fit a linear model to the arrays (see methods). An adjusted p-value < = 0.05 was employed as a significance threshold. 7 and 577 unam- biguous cDNAs were found to be up-regulated in heart and skeletal muscle respectively (table 3 and additional file 12; columns f and g, both heart and muscle sheets). Combined, this represented 196 non-redundant genes, of which 62 were predicted to be angiogenic (table 3 and additional file 12; columns f and g, both heart and muscle sheets). Differential gene expression on initial cooling: overlap with ratio subtraction and Limma analyses Both these methods are seeking to find genes up-regulated at the first time point on immediate cooling of fish vs. fish that have acclimatised to the temperature. The results should corroborate each other. Dealing the heart tissue first, there were 7 cDNAs that reached a p-value < = 0.5 and 3 cDNAs that had a ratio subtraction > = 2 (see addi- tional file 12, sheet 12a). These cDNAs did not overlap but the ratio subtraction results, though not significant, for the Limma cDNAs were positive. So they concurred but not with significance for the ratio subtraction. Muscle, in contrast, gave better corroboration between ratio and Limma analyses. There were 577 cDNAs with Limma adjusted p-value < = 0.05. There were 82 total ratio subtractions that reached significance (see additional file 12, sheet 12b). Of these, 47 agreed with significant Limma results. The results were encouraging. In general, the number of genes predicted by a method are always a bal- ance between false positives and false negatives. In this case 47 cDNAs are in common in total between the "ratio" and the "Limma" methods for muscle. However, differ- ences between the two methods are expected as the first is based on a hard cut-off on the fold change, the second is based on a conservative cut-off on a p-value after multiple test correction (so it accounts for the variation, not only for the mean value; and also for multiple tests). The rea- son for using the different methods is: if 3 biological rep- licates were present than the second would be the ideal, however, here only different temperatures were used as Page 9 of 17 (page number not for citation purposes)