Urzúa et al. BMC Genomics 2010, 11(Suppl 5):S1 http://www.biomedcentral.com/1471-2164/11/S5/S1 Page 4 of 11 Table 2 Summary of correlation results between ovarian gene expression and phenotypes Trait Correlated clonesa Trait interactionsb Direction and strength of correlation (R value range)c Gene expression shift (δ log2 value range)d Ovarian tumor frequency (OTF) Number of litters (NL) Litter size (LS) Relative fecundity (RF) 425 (280) 234 (82) 73 (66) 17 (3) OTF NL LS RF - 145 0 0 145 - 0 0 0 7 0 7 - 7 7 - Positive 97 (1.00-0.51) 161 (1.00-0.73) 53 (0.99-0.78) 12 (0.98-0.78) Negative 328 (1.00-0.52) 72 (0.99-0.67) 20 (0.99-0.62) 5 (0.98-0.90) Positive Negative 3.26 - 0.19 2.96 - 0.21 2.94 - 0.32 3.00 - 0.24 2.50 - 0.32 2.18 - 0.41 1.50 - 0.29 0.85 - 0.51 a Number of clones resulting from a regression analysis with false discovery rate (FDR) control performed with the multiple test tool Pomelo (accessible at http:// pomelo2.bioinfo.cnio.es/). For each trait, genes were filtered with an adjusted p<0.05 resulting in a total of 590 unique arrayed clones showing statistically significant correlation. Under this criterion, the trait “productive matings” did not show correlation. The number of clones exclusively correlated with the indicated trait is shown between parentheses. b Interactions correspond to the number of clones correlated with two or more traits as obtained with Boolean comparisons. c Pearson correlation coefficients (R) were calculated using gene expression log2 ratios as independent variable and each of phenotypic trait (see Table 1) as dependent variables. The observed range of R values for all correlated genes is shown between parentheses. d Gene expression ratios (log2) for the extreme trait values were subtracted (δ) and then ranked. The highest and the lowest ratio difference are shown between parentheses. correlated gene. The gene ontology (GO) profile of the 401 known genes included “regulation of transcription” (67 genes; p= 4.4e-7), “RNA binding” (44 genes; p= 4.3e-16) and “RNA metabolism” (26 genes; p= 1.0e-7) with the highest statistical significance in a hypergeo- metric test. Notably, even though GO terms are inher- ently redundant, the combined 3 functional groups corresponded to 26% (105) of known significant genes. The overall functional profile of the 401 genes list is detailed in Additional file 1, table 3. Ovarian tumor fre- quency (OTF) was the top correlated trait followed by number of litters (NL). The relationship between the squared correlation coefficient (R2) for each of the 425 OTF correlated clones and its corresponding gene- expression shift is depicted in Figure 2. Negative and positive correlation was observed for 328 and 97 clones, respectively. Notably, a large fraction of strongly corre- lated genes (R2 = 0.8 to 1.0) showed gene-expression shifts under 1 unit in the log2 ratio scale (mean +0.53 and -0.58 in the yellow highlighted quadrants) which equals to less than 2-fold up- or down-regulation in the linear scale across the 4 strains. Selected clones either with high significance, large expression shift or both are tagged with red and green dots in Figure 2. OTF positively correlated genes included the unknown function H3055D10 clone, the BC003993 clone coding for the KIAA1604 protein presumably implicated in nuclear mRNA splicing [18], the heat shock proteins Hspb1, Hsp90aa1 and Dnajb1 involved in protein fold- ing and cellular stress [19], and the gene Star (steroi- dogenic acute regulatory protein), which mediates mitochondrial cholesterol transport for its conversion to pregnenolone [20]. On the other hand, genes nega- tively correlated with OTF included Ogt, coding for a N-acetylglucosamine transferase enzyme activity impli- cated in heat-stress response [21], the mRNA splicing genes Hnrnpa2b1, a possible early detection marker of lung cancer [22], and Sfrs5 which is overexpressed in breast tumors [23]. Additional OTF(-) genes included 2310043N10Rik corresponding to a virus-inducible non-coding RNA (VINC) expressed in brain and sev- eral adult non-neuronal mouse tissues [24], the two clones of Malat1, a long, non-coding metastasis-asso- ciated lung cancer transcript up-regulated various tumors including ovarian cancer [25], and Clk1 (CDC- like kinase 1), involved in nuclear phosphorylation of serine/arginine-rich proteins in the spliceosomal complex [26]. Figure 2 Strength of OTF correlation as a function of ovarian gene expression shift The squared correlation coefficient (R2) for the 425 clones positively and negatively correlated (adjusted p<0.05) with ovarian tumor frequency (OTF) were plotted against their respective gene expression shifts. The shift is defined as the resultant of the subtracted gene expression log2 ratios between extreme values (see Table 1 and Additional file 1, 2, 3 Results file).