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

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:In this work we aimed at sequencing and assembling the goat milk transcriptome corresponding at colostrum and 120 days of lactation. To reconstruct transcripts we used both the genome as reference, and a de novo assembly approach. Additionally, we aimed at identifying the differentially expressed genes (DEGs) between the two lactation stages and at analyzing the expression of genes involved in oligosaccharides metabolism.A total of 44,635 different transcripts, organized in 33,757 tentative genes, were obtained using the goat genome as reference. A significant sequence similarity match was found for 40,353 transcripts (90%) against the NCBI NT and for 35,701 (80%) against the NR databases. 68% and 69% of the de novo assembled transcripts, in colostrum and 120 days of lactation samples respectively, have a significant match with the merged transcriptome obtained using Cufflinks/Cuffmerge. CSN2, PAEP, CSN1S2, CSN3, LALBA, TPT1, FTH1, M-SAA3, SPP1, GLYCAM1, EEF1A1, CTSD, FASN, RPS29, CSN1S1, KRT19 and CHEK1 were found between the top fifteen highly expressed genes. 418 loci were differentially expressed between lactation stages, among which 207 and 122 were significantly up- and down-regulated in colostrum, respectively. Functional annotation and pathway enrichment analysis showed that in goat colostrum somatic cells predominate biological processes involved in glycolysis, carbohydrate metabolism, defense response, cytokine activity, regulation of cell proliferation and cell death, vasculature development, while in mature milk, biological process associated with positive regulation of lymphocyte activation and anatomical structure morphogenesis are enriched. The analysis of 144 different oligosaccharide metabolism-related genes showed that most of these (64%) were more expressed in colostrum than in mature milk, with eight expressed at very high levels (SLCA3, GMSD, NME2, SLC2A1, B4GALT1, B3GNT2, NANS, HEXB).To our knowledge, this is the first study comparing goat transcriptome of two lactation stages: colostrum and 120 days. Our findings suggest putative differences of expression between stages and can be envisioned as a base for further research in the topic. Moreover because a higher expression of genes involved in immune defense response, carbohydrate metabolism and related to oligosaccharide metabolism was identified in colostrum we here corroborate the potential of goat milk as a natural source of lactose-derived oligosaccharides and for the development of functional foods.

Project description:Even in genomes lacking operons, a gene's position in the genome influences its potential for expression. The mechanisms by which adjacent genes are co-expressed are still not completely understood. Using lactation and the mammary gland as a model system, we explore the hypothesis that chromatin state contributes to the co-regulation of gene neighborhoods. The mammary gland represents a unique evolutionary model, due to its recent appearance, in the context of vertebrate genomes. An understanding of how the mammary gland is regulated to produce milk is also of biomedical and agricultural importance for human lactation and dairying. Here, we integrate epigenomic and transcriptomic data to develop a comprehensive regulatory model. Neighborhoods of mammary-expressed genes were determined using expression data derived from pregnant and lactating mice and a neighborhood scoring tool, G-NEST. Regions of open and closed chromatin were identified by ChIP-Seq of histone modifications H3K36me3, H3K4me2, and H3K27me3 in the mouse mammary gland and liver tissue during lactation. We found that neighborhoods of genes in regions of uniquely active chromatin in the lactating mammary gland, compared with liver tissue, were extremely rare. Rather, genes in most neighborhoods were suppressed during lactation as reflected in their expression levels and their location in regions of silenced chromatin. Chromatin silencing was largely shared between the liver and mammary gland during lactation, and what distinguished the mammary gland was mainly a small tissue-specific repertoire of isolated, expressed genes. These findings suggest that an advantage of the neighborhood organization is in the collective repression of groups of genes via a shared mechanism of chromatin repression. Genes essential to the mammary gland's uniqueness are isolated from neighbors, and likely have less tolerance for variation in expression, properties they share with genes responsible for an organism's survival.

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Sequencing the transcriptome of milk production: milk trumps mammary tissue.

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