Wickramasinghe et al. BMC Genomics 2012, 13:45 http://www.biomedcentral.com/1471-2164/13/45 Table 4 Expression values of genes encoding enzymes in ubiquitin-proteasome pathway at 15, 90 and 250 days of lactation (Continued) PSMD2 PSMB2 41.15 39.62 82.81 84.58 112.20 115.62 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 with highest expression observed in USP10 at late lacta- tion. All the genes, except VCPIP1, showed higher expression in late lactation. VCPIP1 had the highest expression in peak lactation. The overall expression pattern of genes in UPP showed a higher expression of most of the genes along the course of lactation. This expression pattern agrees with pathway analysis results of the current study and the observation of Lemay et al. in early involuting mouse mammary gland [31] and it suggests a similar pattern of cellular protein degradation in mouse and cow with highest protein turn over occurring at later stages of lactation. Conclusions This is the first published study on the global expression profiling of genes in the somatic cells of milk of any mammalian species. Sixty nine percent of genes anno- tated in NCBI Btau 4.0 bovine genome assembly were expressed in somatic cells. There was ubiquitous expres- sion of ~9,000 genes while ~6,930 genes had a signifi- cant change in expression with the stage of lactation. The highest number of genes were expressed in peak lactation (day 90) MSC. Genes encoding caseins, whey proteins and enzymes in the lactose synthesis pathway showed high expression in transition lactation (day 15) MSC, and indicated higher production of casein and whey derived bio-active peptides. Most of the genes in fat metabolism also had high expression in transition and peak lactation MSC. There was an increase in the expression of genes in UPP along the course of lacta- tion. Most of the endogenous milk proteases were expressed in peak and late lactation MSC and the important findings obtained from the detailed analysis of protease gene expression highlight the significance of metabolic pathway-based gene expression analysis. Ana- lysis of the results obtained on all the gene network pathways are beyond the scope of this article and this is the first chapter of a fascinating journey on the biology of milk and milk somatic cells. Methods Animals and their management Six healthy mastitis-free Holstein cows in their second or third lactation at the UC Davis dairy were selected Page 11 of 14 for the study. The animals were kept in free stall hous- ing, fed total mixed ration (TMR) and had access to water ad libitum. Cows were milked twice daily, at 4 a. m. and 4 p.m., in the milking parlor and managed according to AAALAC (American Association for Accreditation of Laboratory Animal Care) guidelines. Sample collection was approved by the UC Davis Insti- tutional Animal Care and Use Committee (IACUC) pro- tocol #16151. Microscopic examination with a trypan blue exclusion test for viability of milk somatic cells [55] was conducted in a preliminary study. Milk samples were collected from 4 cows (150-170 day lactation) at 0, 1, 2, 3 and 4 hours after the morning milking and exam- ined under the microscope. The same procedure was repeated in another set of 4 cows in a separate day. The highest percentage of epithelial cells (11.6%) and the highest viability (86%) were detected 3 hours after morning milking. Therefore fresh milk samples were collected by hand milking the four quarters of the cows (50 ml from each quarter) at days 15, 90 and 250 of lac- tation 3 hours after the morning milking (three biologi- cal replicates per stage of lactation with a total of 9 samples). Initially the study was designed to analyze the milk obtained at days 15 and 250 and later, milk obtained at day 90 was included in the study. Therefore the day 15 and day 250 samples were collected from the same three cows whereas the day 90 samples were col- lected from three different cows. Day 90 cows were well matched to the other ones. Day 90 cows were selected from the same dairy and the animals were kept under the same management conditions. One animal collected at days 15 and 250 was in the third lactation, while the other two animals in these collection days were in their second lactation. Therefore, at day 90, one animal sam- ple was collected from a cow in her third lactation and the other two samples were from cows in their second lactation. RNA extraction Milk samples collected from the four quarters were mixed and a sub sample of 50 ml was used for RNA extraction from MSC. Milk cells were pelleted by adding 50 μl of 0.5 M EDTA to 50 ml of fresh milk and centri- fuging at 1800 rpm at 4°C for 10 minutes. The pellet of cells was washed with 10 ml of PBS at pH7.2 and 0.5 mM EDTA and filtered through a sterile cheese cloth to remove any debris. Milk cells were then centrifuged again at 1800 rpm, 4°C for 10 minutes. The supernatant was decanted, and RNA was extracted from the milk cell pellet by the Trizol method (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. RNA was quantified by an ND-1000 spectrophotometer (Fisher Thermo, Wilmington, MA), and the quality and integrity was assessed by the spectrophotometer 260/