Wickramasinghe et al. BMC Genomics 2012, 13:45 http://www.biomedcentral.com/1471-2164/13/45 INSIG1 and SCAP genes had highest expression in peak lactation MSC. Expression of endogenous proteases in bovine milk somatic cells Important characteristics of dairy products, such as fla- vor, texture and longevity, are dependent mainly on pro- teins and fatty acids in milk [46]. Therefore, the endogenous proteases have an effect on the physico- chemical characteristics of fresh milk and on the quality of dairy products such as yogurt and cheese [47]. Enzy- matic activities of proteases such as plasmin [48], cathe- psin D [49] and cathepsin B [50] are identified in the bovine milk by biochemical and immunological assays. However, no studies have been conducted on the expression level of the genes encoding these endogenous proteases. The RNA-Seq expression analysis showed significant expression of cathepsin B, D, Z, H and S in the somatic cells of Holstein cow milk at days 15, 90 and 250 of lac- tation (Table 3). Except for cathepsin A and L1, all the other cathepsin genes showed an increase in expression during the course of lactation. This is the first time that expression of cathepsin genes has been measured in a mammalian milk sample, and the high expression of Page 9 of 14 most of the cathepsin genes may indicate the presence of a high enzymatic activity of cathepsins in bovine milk. The plasminogen (PLG) gene was not expressed in any of the MSC samples. Previous studies have shown PLG as not being expressed in the bovine mammary gland [51], and the results of the present study confirm this also for MSC. The origin of plasmin and plasminogen is thought to be by migration from blood to the milk [52]. The lack of expression of PLG in MSC agrees with this migration theory of plasminogen. However, the other genes involved in the plasmin system were expressed in the MSC (Table 3). Urokinase type plasminogen activa- tor (PLAU), urokinase receptor (PLAUR) and the plas- minogen activator inhibitor (SERPINE1) had increased expression in peak lactation. These results indicate an active conversion of migrated plasminogen to plasmin in bovine milk and higher activity of plasmin in peak lacta- tion milk. Even though elastase is a principal protease in neutrophils, there is no published literature on the enzy- matic activity of elastase in milk. The elastase-1 gene showed expression in bovine MSC with the highest expression in peak lactation milk samples. The gene expression analysis showed lower expression of proteases in transition lactation when compared to peak and late lactation MSC. Late lactation had the Table 3 Expression values of genes encoding endogenous proteases in milk somatic cells at 15, 90 and 250 days of lactation Gene symbol1 Day 152 Day 902 Day 2502 1Gene symbol according to NCBI Entrez gene database http://www.ncbi.nlm.nih.gov/gene/ 2Days 15, 90 and 250 gene expressions given in RPKM Category Cathepsins Elastase Plasminogen pathway Enzyme Cathepsin B Cathepsin D Cathepsin Z Cathepsin H Cathepsin S Cathepsin C Cathepsin K Cathepsin A Cathepsin F Cathepsin W Cathepsin L2 Cathepsin O Cathepsin L1 Cathepsin G Elastase 1 Elastase 3B Plasminogen Urokinase-typeplasminogen activator Urokinase receptor Tissue plasminogen activator Plasminogen activator inhibitor Plasmin inhibitor CTSB CTSD CTSZ CTSH CTSS CTSC CTSK CTSA CTSF CTSW CTSL2 CTSO CTSL1 CTSG ELA1 ELA3B PLG PLAU PLAUR PLAT SERPINE1 SERPINF2 1062.9 786.0 222.0 122.9 89.1 69.3 82.1 86.8 22.1 16.7 10.5 0.8 0.9 0.0 0.4 0.0 0.0 18.7 60.4 1.9 3.6 0.0 4463.3 4007.6 1048.3 536.0 507.2 378.1 370.5 432.5 76.2 25.3 12.5 3.7 0.8 0.0 3.2 0.1 0.0 216.8 346.1 4.2 79.7 0.3 6052.5 4645.6 1034.0 649.1 642.9 446.4 389.8 334.0 103.3 32.2 13.0 4.4 0.1 0.0 1.2 0.1 0.0 52.2 91.3 4.8 19.5 0.1