Lee et al. BMC Genomics 2010, 11:623 http://www.biomedcentral.com/1471-2164/11/623 Page 3 of 11 No of Genes in whole set: 24,128 No of Genes in present set: 12,745 Table 2 Summary of annotated probes on the bovine genome array Gene annotation Unidentified probe set (unknown genes) 2170 Annotated probe set 21958 Bovine gene transcript annotated by Bovine genome sequence 9897 (45.07%) Predicted genes defined by similarity in the bovine species 7275 (33.13%) Transcripts defined by similarity in other species (human and mice) 4786 (21.79%) The correlation of fold changes in gene expression between the arrays and PCR is shown in Figure 2. The results demonstrate a consistent gene expression pattern between both methods. Gene Ontology (GO) and pathway analysis DE genes were analyzed in the context of their GO biolo- gical process. Due to the incomplete annotation of the bovine genome, only 14 out of 21 differentially expressed probe sets were annotated. Top ranking biological GO terms are listed in Table 4, together with the genes asso- ciated to the GO term. The pathway Studio v6.0 program (Ariadne Genomics, Inc) was used to identify molecular connections between the proteins encoded by the 14 annotated differentially expressed genes. The program searches through the ResNet database for all known interactions between genes/proteins such as physical interactions and Figure 1 Distribution of 136 differentially expressed genes across three different data-processing methods (MAS5.0; 65, RMA; 37 and GCRMA; 34). The 21 differentially expressed genes were defined as those detected in at least two data processing methods. 561 12184 5677 (46.59%) 4232 (34.73%) 2275 (18.67%) regulation of expression (Figure 3). Out of the 14 anno- tated genes, 5 main pathway “hubs” (SH3KBP1, THOP1, ADAMTS4, CYP51A and SQLE) were detected in the pathway analysis. The CYP51A and SQLE proteins that are up-regulated in highly marbled muscle appear to be involved in steroid biosynthesis and cholesterol metabo- lism. In particular, CYP51A is activated by two proteins, SP1 and SREBP1 which are common transcription factors in lipid metabolism. SH3KBP1 is involved in cell pro- cesses such as intracellular signaling cascade, oxidative stress and cell proliferation. In addition, the pathway ana- lysis demonstrated that ADAMTS4 is activated by immune responses related to single molecules (IL-17A, TNF, NF-kB and IL-1 family) and transforming growth factor beta 1 (TGFb1). The results suggest a biological pathway connecting CYP51A, SQLE and ADAMTS4 that has not been previously identified in bovine gene expres- sion studies on marbling fat. Gene expression of selected genes in the ADAMST4 pathway GO and pathway analysis showed that ADAMTS4 is involved in protein catabolic process (GO0006516) and it is mainly activated by the immune related single molecules IL-17A, TNFa and TGFb1. The ADAMTS4 gene has a role in proteolysis degradation of the extra- cellular matrix (connective tissue) in muscle. Marbling fat accumulates in the connective tissue matrix in close proximity to blood vessels. These findings suggest that ADAMTS4 might be involved in a pathway associated to phenotypic differences of marbling fat in cattle. To determine if any of the three regulators IL-17A, TNFa and TGFb1 were associated with the ADAMTS4 pathway, we investigated gene expression of ADAMTS4 and the three regulators (IL-17A, TNFa and TGFb1) in muscle with divergent IMF and protein content using RT- PCR. As shown in Figure 4, expression of ADAMTS4 increases significantly as intramuscular fat content increases (P = 0.01) and muscle protein content decreases (P = 0.01). Of the regulators only TGFb1 significantly increased expression with increasing intramuscular fat content (P = 0.03) and it tended to decrease with decreas- ing muscle protein content (P = 0.08). In summary, TGFb1 and ADAMST4 are highly associated with increases in marbling fat.