Lee et al. BMC Genomics 2010, 11:623 http://www.biomedcentral.com/1471-2164/11/623 number of complicated biological pathways which are related to adenoreceptor signaling [5], the cytokine sig- naling pathway [6] and a wide range of hormonal and transcriptional factors such as leptin [7], adiponectin [8] and insulin like growth factor protein families [9]. As such, this interaction between muscle and fat also reflects a biochemical signaling pathway within the mus- cle. Therefore, marbling differences might be a function of a series of complex interactions between biological pathways [10,11]. The completion of the bovine genome project pro- vided a tool for genome-wide functional studies to understand the interactions of complex biochemical pathways involved in protein and fat synthesis. For example, Affymetrix produces an oligonucleotide Bovine Genome Array that allows genome wide global profiling of over 23,000 bovine transcripts simultaneously Micro- array based gene expression analyses associated with beef meat quality have focused on detecting differen- tially expressed genes in different breeds [12] and differ- ent nutritional treatments [13]. However, there is no report on gene expression differences in muscle with divergent marbling phenotypes within breed [14]. Here we report the results of a study undertaken to identify the biochemical differences in m. longissimus with divergent marbling phenotypes. The objective of this study was to identify differentially expressed genes and their role in a signaling pathway in m. longissimus with a wide range of marbling phenotypes. Results Differentially expressed genes between high and low marbling muscle To detect the relationship between differentially expressed genes with marbling score, samples from m. longissimus were taken from ten unrelated animals with the highest and lowest marbling score. It was recognized that given the divergent nature of the animals the rela- tionship would be inflated above that found in a nor- mally sampled population. Table 1 shows the summary statistics for the ten ani- mals used in this microarray analysis. Intramuscular fat content (IMF) values range from 4% to 32% in the cho- sen animals with a clear contrast between the high and low marbled groups. After hybridization with the Affy- metrix bovine genome array, gene expression intensities were measured using three pre-processing methods: MAS5.0, RMA and GCRMA. Table 2 shows the summary of annotated probe sets on the bovine genome array. The array contains 24,128 probe sets, 91% of which were annotated by the manu- facturer. Out of these, 12,745 (50%) were consistently detected as “Present (P)” in all samples according to the MAS5.0 background correction algorithm. Of the 12,745 Page 2 of 11 Table 1 Summary statistics for marbling score, intramuscular fat and protein percentage for the low and high divergent marbling groups used for the gene expression analysis Groups Animal Marbling score (range 1-7) IMF (%) Protein (%) 7.11 6.02 4.88 7.36 12.04 24.35 32.49 16.56 26.24 18.81 21.07 21.66 21.30 21.05 21.23 16.06 15.96 17.27 17.22 18.07 Low High 509 537 554 670 691 527 547 586 589 632 2 2 3 3 3 7 7 7 7 7 probes in the P set, 12,184 have well annotated informa- tion (Table 2). A moderated t-test using Limma (33) was used to explore genes differentially expressed between high and low marbled animals in m. longissimus., We found 136 differentially expressed genes between high and low- marbled muscle using three data processing methods: MAS5.0 (65 transcripts), RMA (37 transcripts) and GCRMA (28 transcripts) (Figure 1). Twenty one genes showed significant differential expression (P < 0.01) in at least two normalization methods (Table 3). Out of the 21 differentially expressed genes (DEGs) listed in Table 3, eight differentially expressed genes were identified as up-regulated in mus- cle with high intramuscular fat content and the remain- ing 13 DEGs were down-regulated in samples with high intramuscular fat content. Putative genes were assigned to 14 out of the 21 DEGs (Table 3). The remaining 7 DEGs match gene sequences in bovine and other species with strong, moderate or weak sequence similarity but have no functional annotation assigned (Table 3). Based on the gene identities and associated functions, three up-regulated genes are involved in lipid metabolism (squalene epoxidase and cytochrome P450) and muscle metabolism (SH3 domain YSC-like 1). Three down- regulated genes (ATP binding protein, Proteasome acti- vator subunit 4 and Thimetoligopeptidase 1) belong to functional classes involved in energy metabolism and an intracellular metabolic pathway. The remaining 7 DEGs are only hypothetical proteins or transcribed loci (single EST clones). Out of these 7 DEGs, three hypothetical proteins (LOC788205, LOC509649 and LOC777601) and two transcribed loci (Bt.19107.2.A1_at and Bt.19107.1. S1) could be worth pursuing further to elucidate their functional role in marbling. Technical validation of the 21 differentially expressed genes from the microar- ray experiment was confirmed by real-time PCR.