UC San Diego

Human milk is an important biofluid for nutrition and regulatory signaling transmitted from mother to infant and is also known to be rich in microRNA (miRNA), a small noncoding RNA that can prevent translation or promote mRNA degradation. miRNA can be found inside extracellular vesicles (EVs), that likely protect them from digestion and RNase degradation in an infant’s stomach. Thus, these miRNAs represent a potential for intergenerational transmission of epigenetic signals from mother to infant. Little research has investigated miRNA activity in human milk, and no consensus exists on the best technique for isolating EVs in milk for the eventual goal of small RNA sequencing. The objective of this study is to compare four techniques for isolating EVs from human milk for miRNA analyses: 1) Exoquick, 2) ultra-centrifugation, 3) ExoRNeasy and 4) Size Exclusion Chromatography (SEC). An additional goal for the SEC approach is to determine which fractions of the milk are enriched in EVs to guide the selection and pooling of fractions for downstream miRNA sequencing. Our findings confirm the presence and successful detection of EVs isolated using the Exoquick, ultra-centrifugation and SEC methods. Also, the results identify which SEC fractions likely contain the most EVs. Finally, the study displays the variability of amount and size of small RNA extracted across the methodologies of interest. These results provide a significant foundation to understanding variation among EV-enriched isolates across four distinct methodologies.