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:BACKGROUND: Epigenetic processes enable the environment to modulate gene transcription during fetal development, ultimately leading to stable phenotypic changes throughout the lifespan. There is a relatively high prevalence of altered sleep patterns during late gestation, especially obstructive sleep apnea (OSA). Intermittent hypoxia (IH) is a hallmark of OSA caused by periodic obstructions of the upper airway, which occurs along with sleep fragmentation (SF). We have previously shown that adult offspring of late gestational SF (LG-SF) mothers exhibited metabolic alterations via epigenomic alterations. The aims of this study were to investigate the effects of LG-IH on metabolic features in offspring and to determine the effects of LG-IH on the epigenome of visceral white adipose tissue (VWAT) in the offspring. METHODS: Time-pregnant mice were exposed to LG-IH or room air (RA) paradigms conditions during the last 6 days of gestation (LG-IH and LG-RA groups, respectively). Offspring were weaned at 21 days and fed with normal chow diet. At 24 weeks, lipid profiles and metabolic parameters were assessed. We performed large scale DNA methylation analysis using MeDIP coupled to microarrays (MeDIP-chip) in offspring VWAT (n=8 mice per group). Regions exhibiting differential DNA methylation (DMRs) between the groups were mapped to their corresponding genes and tested for potential overlaps with biological processes and pathways using Ingenuity Pathway Analysis. RESULTS: We detected 1520 DMRs between the LG-IH and LG-RA groups (p< 0.0001, two-way ANOVA), associated with 693 genes. A significant portion of these genes (n=162) were also associated with previously identified DMRs in LG-SF offspring (p=1.35 x 10-97, hypergeometric test). Pathway analyses showed that genes affected by LG-IH and LG-SF, were mainly associated with molecular processes related to metabolic regulation and inflammatory response. In particular for LG-IH, biochemical pathways related to energy production through amino acid and fatty acid metabolism (i.e. methionine, choline and 2-oxobutanoate degradation pathways, methylmalonyl pathway, cysteine biosynthesis, etc.). CONCLUSIONS: Our findings suggest a major role for epigenomic alterations in the metabolic dysfunction of adult offspring born to LG-IH and LH-SF mothers. Furthermore, whereas LG-IH may concomitantly act upon metabolic pathways also affected by LG-SF, LG-IH also affects biochemical processes underlying energy production in VWAT of the offspring.

Dataset Information

Investigation of Age Gelation in UHT Milk.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure