Wickramasinghe et al. BMC Genomics 2012, 13:45 http://www.biomedcentral.com/1471-2164/13/45 different groups of genes in MSC along the course of lactation. Milk component related genes show higher expression in early lactation while the expression of apoptosis related genes increased toward the end of lac- tation. These suggest that the MSC are viable, transcrip- tionally active cells that can adapt to the conditions and needs of the lactation stage. In order to examine the global functional changes associated with the expression of genes during lactation, all highly expressed genes (≥ 500 RPKM) at each stage of lactation were classified using the gene ontology (GO) terminology using the BLAST2GO software portal [28]. Highly expressed genes in three stages of lactation showed similar enrichment in most of the biological process, molecular function and cellular component GO terms. However, some GO terms showed differences in enrichment with the lactation stage. One important observation was the progressive increase in biological process GO terms for the immune process and biologi- cal regulation along the course of lactation (Additional File 1, Figure S1). Molecular function GO terms for electron carrier activity and proteosome regulator activ- ity were detected only in peak and late lactation MSC samples. These results agree with previous gene expres- sion studies conducted in the mouse mammary gland where immune related genes showed increased expres- sion toward the later stages of lactation [29]. It is thought that immune cells are involved in defense, cyto- kine signaling and phagocytosis in the involuting mam- mary gland [29]. Increased immune cell function can lead to high protein turn over and this is reflected in enrichment of proteasome regulator GO term toward late lactation. Another important observation was that compared to the two other stages, the highly expressed genes in transition lactation MSC had a relatively high percentage of genes encoding proteins in non-mem- brane bound organelle locations. Over all analysis of GO annotations showed that most of the genes expressed in MSC have similar functions and locations regardless of the lactation stage. However a fraction of the milk transcriptome has genes devoted to specific functions unique to the lactation stage, and proteins encoded by these genes function in specific cel- lular locations. Global analysis of genes with statistically significant changes in expression Approximately 9,000 genes (53%) showed ubiquitous expression in bovine MSC. In order to identify the sig- nificant metabolic pathways represented by the genes with ubiquitous expression, a pathway analysis was con- ducted using the MetaCore portal (GeneGo, a Thomson Reuters business). Metabolic pathways represented by the genes with ubiquitous expression showed the Page 5 of 14 involvement of most of these genes in housekeeping processes such as energy generation and cytoskeleton remodeling. The complete list of pathways and their p values are provided in the Additional File 2, Table S1. The highest number of genes (6,243 genes) with sig- nificant expression differences (p ≤ 0.05, FDR q ≤ 0.3) was found between transition and peak lactation MSC. Among these genes 1,539 (24%) had higher expression in transition lactation MSC and 4,704 (76%) had higher expression in peak lactation MSC. BLAT2GO analysis conducted to study the functional changes associated with up regulated genes showed similar enrichment of GO terms in transition and late lactation. In agreement with observations in the previous section, the cells death biological process GO term and proteasome regulator molecular function GO term was detected only in peak lactation. MetaCore pathway analysis of significantly up regu- lated genes at transition and peak lactation showed Phosphatidylinositol (3, 4, 5)-trisphosphate (PIP3) sig- naling pathway to have the most significant p value (p = 4.22 × 10-5) for enrichment in up-regulated genes in transition lactation MSC. PIP3 signaling activates the protein kinase Akt that regulates cell growth, prolifera- tion and survival [30]. Lemay et al [31] observed the PI3-Akt pathway to be highly significant during lactation in mouse mammary gland. Therefore, our results agree with the observations of mouse mammary gland gene expressions, and the enrichment of PIP3 pathway in up regulated genes of transition lactation indicates an enhanced cell growth and proliferation in transition lac- tation compared to peak lactation. Cytoskeleton remo- deling pathway had the most significant p value (p = 1.25 × 10-13) for enrichment for up regulated genes in peak lactation. However, the cytoskeleton remodeling pathway was also present in the 10 most significant pathways in transition lactation. There were several pathways that showed significant enrichment in both transition and peak MSC samples and this indicates that even though there are different sets of up regulated genes at two stages of lactation; most of these up regu- lated genes are involved in regulating the same meta- bolic pathways. The complete list of significant pathways and their p values are provided in the Addi- tional File 3, Table S2. There were 5,218 genes with statistically significant expression differences (p ≤ 0.05, FDR q ≤ 0.3) between transition and late lactation MSC. Among these genes, 1,257 (24%) had higher expression in transition lactation milk and 3,961 (76%) had higher expression in late lac- tation milk. BLAST2GO analysis showed similar enrich- ment of most of the GO terms in the up regulated genes at two stages of lactation. As expected, the cell death and proteasome regulator GO terms were only