Wickramasinghe et al. BMC Genomics 2012, 13:45 http://www.biomedcentral.com/1471-2164/13/45 lower than in human milk oligosaccharides [9]. The majority of these free oligosaccharides in bovine milk are sialylated whilst in human milk most of them are fucosylated [10]. Because of these differences between human and bovine milk, it would be desirable to change the composition of cow milk according to specific needs of target groups such as infants or immune compro- mised individuals. In order to achieve this goal, a thor- ough understanding of the components and the regulation of bovine milk composition is required. Cow milk contains a heterogeneous population of somatic cells consisting of lymphocytes, neutrophils, macrophages and exfoliated epithelial cells [11]. These cells are responsible for the synthesis and secretion of components such as proteins, lipids and oligosaccharides in to the milk [12,13]. Even though many studies have been conducted on the physicochemical properties of cow milk and the genes expressed in bovine mammary gland [11,14], limited research has been published on the detailed characterization of genes expressed in somatic cells in milk. In a previous study we identified extensive similarities between the mammary gland and milk somatic cell transcriptome of the same cow [15]. Most of the genes expressed in the mammary gland transcriptome were present in milk somatic cells (MSC). Compared with the mammary gland, higher numbers of genes were expressed in MSC. Sets of genes related to immunity, organ development and behavior were uniquely expressed in MSC. Therefore, the identification and characterization of genes expressed in MSC repre- sent an important step toward understanding the com- plex biological properties and species-specific variations of milk. In the current study we used RNA sequencing (RNA- Seq) technology to examine the genes expressed in tran- sition (day 15), peak (day 90) and late (day 250) lacta- tion somatic cells in Holstein cow milk. Day 15 was selected to study the transition occurring from early lac- tation to peak lactation. Day 90 represents the peak lac- tation stage with the highest milk production. Day 250 represents the milk produced in the involuting mam- mary gland (in the initial stage). A global analysis was conducted first on the bovine milk transcriptome by studying the highly expressed genes in each stage of lac- tation and genes with statistically significant expression between the stages. Then a detailed analysis was con- ducted on the expression of important genes encoding enzymes in major milk component synthesis pathways (lactose, protein and fat), endogenous proteases and enzymes in ubiquitin- proteasome pathway. Figure 1 shows the analytical flow chart that was followed in the study. Page 2 of 14 Figure 1 Analytical flowchart to study bovine milk transcriptome by RNA-Seq. Nine milk samples was collected at 15, 90 and 250 days of lactation with three biological replicates per stage. An initial global analysis was conducted on genes expressed in bovine MSC followed by a detailed analysis on genes encoding enzymes in major milk component synthesis pathways, endogenous milk proteases and enzymes in ubiqutin-proteasome pathway. Results and Discussion Global analysis of gene expression in three stages of lactation RNA sequencing produced a total of ~200 million reads with an average of 23 million reads for each stage of lac- tation. Three biological replicates were analyzed for each stage of lactation with the reads ranging from 11-28