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Updated: Sep 5, 2025
Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile
Published on: June 13, 2016
Quantitation of Exosomes and Their MicroRNA Cargos in Frozen Human Milk
Haichuan Wang1, Di Wu2, Sonal Sukreet2
1Department of Nutrition & Health Sciences, University of Nebraska-Lincoln.
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.
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.
