Quantitation of Exosomes and Their MicroRNA Cargos in Frozen Human Milk
Haichuan Wang, Di Wu, Sonal Sukreet, Anthony Delaney, Mandy B. Belfort, Janos Zempleni
Abstract
We assessed feasibility of analyzing exosomes and microRNA cargos in frozen human milk as a prerequisite for epidemiological studies of milk exosomes. We collected milk from 5 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 3 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 × 10 9 ± 3.8 × 10 9 to 5.6 × 10 8 ± 9.7 × 10 8 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.