Dataset Information

Quantitation of Exosomes and Their MicroRNA Cargos in Frozen Human Milk.

ABSTRACT: We assessed feasibility of analyzing exosomes and microRNA cargos in frozen human milk as a pre-requisite for epidemiological studies of milk exosomes. We collected milk from five mother-preterm infant dyads at 3 time points during postnatal hospital care for storage at -80°C. We purified exosomes by ultracentrifugation, probed marker proteins using immunoblots, assessed size and counts with a nanoparticle tracker, and quantified three microRNAs with quantitative PCR. Positive exosome marker proteins were detectable; β-casein was the only detectable contaminant. Exosome count and size trended to decrease from early to late samples (count: 2.3×10⁹ ± 3.8×10⁹ to 5.6×10⁸ ± 9.7×10⁸ exosomes/mL; size: 117 ± 25 to 92 ± 16 nm). Two microRNAs were detectable in early samples only; cycle threshold values equaled 28.7 ± 0.7 for miR-30d-5p and miR-125a-5p; miR-423-5p was not detectable. We conclude that the analysis of exosomes and quantification of microRNAs is feasible in human milk previously stored at -80°C.

SUBMITTER: Wang H

PROVIDER: S-EPMC9258983 | biostudies-literature | 2022 Feb

REPOSITORIES: biostudies-literature

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Quantitation of Exosomes and Their MicroRNA Cargos in Frozen Human Milk.

Wang Haichuan H Wu Di D Sukreet Sonal S Delaney Anthony A Belfort Mandy B MB Zempleni Janos J

JPGN reports 20220201 1

We assessed feasibility of analyzing exosomes and microRNA cargos in frozen human milk as a pre-requisite for epidemiological studies of milk exosomes. We collected milk from five mother-preterm infant dyads at 3 time points during postnatal hospital care for storage at -80°C. We purified exosomes by ultracentrifugation, probed marker proteins using immunoblots, assessed size and counts with a nanoparticle tracker, and quantified three microRNAs with quantitative PCR. Positive exosome marker pro ...[more]

PMID: 35812131

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Project description:BackgroundMilk exosomes are a rich source of microRNAs (miRNAs) that are protected from degradation. Ingestion of milk and subsequent absorption of miRNAs into recipient cells by endocytosis may play a role in the regulation of neonatal innate and adaptive immunity. In contrast, the miRNA content of milk exosomes may also be indicative of a lactating animal's health; whereby, the presence or absence of specific miRNAs could serve as biomarkers for early detection of bacterial infection that can lead to mastitis. In the present study, we therefore analyzed and compared miRNA expression profiles of milk exosomes from four Holstein cows obtained during mid-lactation prior to and after infection (48 h) of the mammary gland with Staphylococcus aureus.MethodsMilk exosomes, purified from control and S. aureus infected cows, were extracted for RNA. Following preparation indexed libraries from both groups the samples were subjected to next generation sequencing.ResultsNext generation sequencing of eight, unpooled small RNA libraries derived from milk exosomes produced about 60.5 million high-quality, bovine-specific sequence reads for comparison of miRNA expression between treatments. Sequence identity analysis showed the miRNAs make up about 13 % of the average RNA content of these exosomes. Although 417 known bovine miRNAs were identified, miRNAs represented the least diverse class of RNA accounting for only 1 % of all unique sequences. The 20 most prevalent unique sequences within this class accounted for about 90 % of the total miRNA-associated reads across samples. Non-annotated, unique reads provided evidence for another 303 previously unknown bovine miRNAs. Expression analyses found 14 known bovine microRNAs significantly differed in frequency between exosomes from infected and control animals.ConclusionsOur survey of miRNA expression from uninfected milk exosomes and those produced in response to infection provides new and comprehensive information supporting a role for delivery into milk of specific miRNAs involved in immune response. In particular, bta-miR-142-5p, and -223 are potential biomarkers for early detection of bacterial infection of the mammary gland. Additionally, 22 mammary-expressed genes involved in regulation of host immune processes and response to inflammation were identified as potential binding targets of the differentially expressed miRNAs.

Dataset Information

Quantitation of Exosomes and Their MicroRNA Cargos in Frozen Human Milk.

Publications

Quantitation of Exosomes and Their MicroRNA Cargos in Frozen Human Milk.

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