Wickramasinghe et al. BMC Genomics 2012, 13:45 http://www.biomedcentral.com/1471-2164/13/45 Table 2 Highly expressed genes in milk somatic cells at 15, 90 and 250 days of lactation Category Highly expressed genes in day 15 Highly expressed genes in day 90 Highly expressed genes in day 250 Gene symbol1 LGB CSN2 CSN1S1 LALBA CSN3 GLYCAM1 CSN1S2 SPP1 CSN2 LGB CSN1S1 FTH1 ACTB CSN3 FTH1 SPP1 CTSB ACTB APOE CTSD CD74 Day 152 Day 902 Day 2502 265631 12515 468 141267 13896 564 73573 11958 423 46445 4187 136 44747 5407 287 25570 3415 124 20952 3116 129 2777 21637 9406 141267 13896 564 265631 12515 468 73573 11958 423 3040 9798 15489 1212 6125 5830 44747 5407 287 3040 2777 1063 1212 1099 786 742 9798 15489 21637 9406 4463 6053 6125 5830 1840 5315 4008 4646 1951 4129 1 Gene symbol according to NCBI Entrez gene database http://www.ncbi.nlm. nih.gov/gene/ 2Days 15, 90 and 250 gene expressions given in RPKM ( -casein), GLYCAM1 (glycosylation dependent cell adhesion molecule-1) and CSN1S2 (casein-a-S2) (Table 2). These genes showed a progressive decrease in expression in peak and late lactation. Except for GLY- CAM1 the other six highly expressed genes encode case- ins and whey proteins. GLYCAM1, a gene in the mucin family, encodes for a milk fat globule glycoprotein. In peak lactation MSC there were 140 (0.7%) highly expressed genes, 5,379 (28%) medium expressed genes and 13,575 genes (71%) with low expression (Table 1). Genes with the highest RPKM values in peak lactation, were SPP1 (secreted phosphoprotein 1), CSN2, LGB, CSN1S1, FTH1 (ferritin heavy chain), ACTB (actin-b) and CSN3 (Table 2). The SPP1 gene encodes for osteo- pontin, a major phosphoprotein involved in bone remo- deling [20]. Previous studies have shown strong associations between the SPP1 gene and bovine milk casein levels. An RNA interference study that depressed the expression of SPP1 gene showed decreased expres- sion of CSN2 and CSN3 in a bovine mammosphere model [21]. It is thought that SPP1 enhances the expres- sion of CSN2 and CSN3 by binding to integrin proteins Page 4 of 14 on mammary epithelial cells [21]. Except for FTH1, the other six highly expressed genes in peak lactation MSC showed decreased expression in late lactation. FTH1 has shown increased expression during mammary gland involution in the mouse [22], and the progressive increase of FTH1 gene expression indicates an involve- ment of FTH1 in mammary gland involution in bovine. FTH1 has been identified as an anti-apoptotic factor [23], and it might be playing a protective role in the involuting bovine mammary gland. ACTB is a major component of the cytoskeleton involved in various types of cell motility functions. ACTB showed the highest expression in peak lactation MSC samples. The changes in expression of ACTB with the stages of lactation indi- cates that, even though ACTB is a popular housekeeping gene [24] that is used for gene expression normalization in many tissues, it cannot be used as a housekeeping gene in MSC over the course of lactation. This was also shown in an experiment conducted by Bionaz and Loor [25] in which they used pair-wise comparison of expres- sion ratios along the course of lactation to predict the best housekeeping genes for the mammary gland. Late lactation MSC had 150 genes (0.8%) with high expression, 5,038 (28%) genes with medium expression and 12,882 (71%) genes with low expression (Table 1). Genes with the highest RPKM values were FTH1, SPP1, CTSB (cathepsin B), ACTB, APOE (apolipoprotein), CTSD (cathepsin D) and CD24 (Table 2). There were remarkable differences between the highly expressed genes in late lactation MSC and genes expressed in transition and peak lactation MSC samples. Casein and whey protein genes were among the highly expressed genes in transition and peak lactation. However, late lac- tation showed high expression of a unique set of genes such as CTSB and CTSD. These genes belong to the cathepsins family, that are proteolytic enzymes found in many tissues, a discussion of genes encoding endogen- ous proteases in milk is presented later in the text. The APOE gene, which codes for Apolipoprotein-E, a lipid transporter with anti-apoptotic activity was also highly expressed [26]. A significant increase in APOE is observed along the course of lactation and, similar to FTH1, APOE may play an anti-apoptotic protective role in the involuting bovine mammary gland. CD74 is expressed in the B-lymphocytes, and the encoded pro- tein promotes the proliferation of B-cells [27]. There- fore, unlike the other two stages of lactation, the highly expressed genes in late lactation MSC are involved mostly in proteolysis, anti-apoptotic activity and immune functions. Previous studies conducted in our laboratory have shown many similarities between the genes expressed in mammary gland and MSC [15]. In the current study we have observed changes in expression patterns of