Project description:Peripheral blood mononuclear cells (PBMC) from cows with experimentally induced hypocalcaemia or spontaneous milk fever were subject of an oligo-microarray analysis following quantitative real-time reverse transcription PCR (q-PCR). In experimental hypocalcaemia induced by 10 % Na2EDTA infusion (n = 4), the microarray revealed that 32 genes were significantly up- or down-regulated compared to control treatment by 11 % CaEDTA infusion (n = 4). In milk fever cases (n = 8), the microarray demonstrated that 98 genes were expressed differentially higher or lower compared to healthy parturient cows (n = 5). From both data, five genes were selected to be strongly related to both experimental hypocalcaemia and milk fever. Additionally, another gene was judged to be specific for milk fever independent of hypocalcaemia. The mRNA expressions of these six genes in milk fever cases were verified by q-PCR, which were significantly higher or lower than those in healthy parturient cows. In the experimental hypocalcemia study, four healthy nonpregnant, non-lactating ovariectomised Holstein cows were used. The experiment was performed according to a 2 M-CM-^W 2 crossover design with 2 weeks intervals. Cows were infused with either Na2EDTA (hypocalcaemic treatment) or CaEDTA (control treatment) solution during a period of 4 h. The animals were monitored for clinical signs of hypocalcaemia (dry nose, staggering, astasia, reduced ruminal activity, increased heart rate, increased respiratory frequency, sweating, and ear cold). Infusion of EDTA solution was temporarily suspended when hypocalcaemia induced astasia was represent. If affected cow stood again, EDTA infusion was resumed. PBMC samples were collected before the start of infusion (0 h; pre-treatment) and at 4 and 24 h after the start of infusion 24h and utilized for gene expression analysis of PBMC. In the spontaneous milk fever study, a total of 13 Holstein dairy cows, which were 8 parturient cows diagnosed with milk fever within 2 days postpartum and 5 clinically healthy parturient cows within 2 days after parturition (Group H), were used. All spontaneous milk fever cases showed hypocalcaemia (< 1.8 mmol/l) in the blood samples obtained before the first treatment, and were treated by an intravenous infusion of a 20 % Ca borogluconate solution (500 ml). The milk fever cases were divided into two categories according to the reponse of the cows to the first Ca treatment: an immediate response in which the cow could stand within 1 day after a single Ca treatment (Group A, n = 4) and a requirement further treatment over 1 day after a single Ca treatment (Group B, n = 4). PBMC samples were collected vein just before the first Ca treatment in groups A and B and within 2 days postpartum in group H and utilized for gene expression analysis of PBMC.

Project description:Studies of normal human mammary gland development and function have mostly relied on cell culture, limited surgical specimens, and rodent models. Although RNA extracted from human milk has been used to assay the mammary transcriptome non-invasively, the transcriptome derived from the milk fat layer has not been compared with the mammary-derived transcriptome nor have sources of RNA been quantified in milk. In this study the effects of milk collection and processing on RNA quality and origin were assessed in humans and rhesus macaques. Total RNA in milk was quantitated in acridine orange-stained milk using an automated whole slide scanner and custom-built Globulator software. Total RNA extracted from milk fat, cells in milk, and mammary biopsies of lactating rhesus macaques were compared using RNA sequencing and analysis. Compared with human milk, milk from macaques contained similar amounts of RNA-containing cytoplasmic crescents, but more cells. Total RNA extracted from milk fractions was also evaluated for factors that affect RNA quality. Degradation of RNA extracted from human milk fat was positively correlated with geographic distance from collection site, storage time, and sample type. There were no differences in RNA degradation in macaque milk collected after 10 min or 4 hr accumulation, suggesting that degradation of RNA extracted from milk fat may not occur in the mammary gland. Using RNA-Seq, RNA extracted from macaque milk fat and cells in milk more accurately represented RNA from mammary epithelial cells (cells that produce milk) than did RNA from mammary tissue. Mammary epithelium-specific transcripts were more abundant in macaque milk fat whereas adipose or stroma-specific transcripts were more abundant in mammary tissue. Functional analyses confirmed the validity of milk as a source of RNA from mammary epithelial cells. Analysis of highly abundant putative microRNAs in macaque milk fat revealed a potentially novel non-coding RNA species that is conserved in humans. RNA extracted from the milk fat during lactation accurately portrayed the RNA profile of milk-producing mammary epithelial cells. However, this sample type clearly requires protocols that minimize RNA degradation.

Project description:Studies of normal human mammary gland development and function have mostly relied on cell culture, limited surgical specimens, and rodent models. Although RNA extracted from human milk has been used to assay the mammary transcriptome non-invasively, the transcriptome derived from the milk fat layer has not been compared with the mammary-derived transcriptome nor have sources of RNA been quantified in milk. In this study the effects of milk collection and processing on RNA quality and origin were assessed in humans and rhesus macaques. Total RNA in milk was quantitated in acridine orange-stained milk using an automated whole slide scanner and custom-built Globulator software. Total RNA extracted from milk fat, cells in milk, and mammary biopsies of lactating rhesus macaques were compared using RNA sequencing and analysis. Compared with human milk, milk from macaques contained similar amounts of RNA-containing cytoplasmic crescents, but more cells. Total RNA extracted from milk fractions was also evaluated for factors that affect RNA quality. Degradation of RNA extracted from human milk fat was positively correlated with geographic distance from collection site, storage time, and sample type. There were no differences in RNA degradation in macaque milk collected after 10 min or 4 hr accumulation, suggesting that degradation of RNA extracted from milk fat may not occur in the mammary gland. Using RNA-Seq, RNA extracted from macaque milk fat and cells in milk more accurately represented RNA from mammary epithelial cells (cells that produce milk) than did RNA from mammary tissue. Mammary epithelium-specific transcripts were more abundant in macaque milk fat whereas adipose or stroma-specific transcripts were more abundant in mammary tissue. Functional analyses confirmed the validity of milk as a source of RNA from mammary epithelial cells. Analysis of highly abundant putative microRNAs in macaque milk fat revealed a potentially novel non-coding RNA species that is conserved in humans. RNA extracted from the milk fat during lactation accurately portrayed the RNA profile of milk-producing mammary epithelial cells. However, this sample type clearly requires protocols that minimize RNA degradation. Transcript profiles from milk cells, milk fat, and mammary tissue from 6 lactating rhesus macaques at 30 and 90 days lactation; 34 samples run in triplicate

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Effect of milk intake on circulating PBMC miRNAs of adult humans

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