Comparison of methods for milk pre-processing, exosome isolation, and RNA extraction in bovine and human milk

Milk is a highly complex, heterogeneous biological fluid that contains bioactive, membrane-bound extracellular vesicles called exosomes. Characterization of milk-derived exosomes (MDEs) is challenging due to the lack of standardized methods that are currently being used for milk pre-processing, exosome isolation, and RNA extraction. In this study, we tested: 1) three pre-processing methods to remove cream, fat, and casein proteins from bovine milk to determine whether pre-processing of whole milk, prior to long-term storage, improves MDE isolations, 2) two commonly-used exosome isolation methods, and 3) four extraction protocols for obtaining high quality MDE RNA from bovine and human milk. MDEs were characterized via Transmission Electron Microscopy (TEM) and Nanoparticle Tracking Analysis (NTA). We also present an optimized method of TEM sample preparation and isolation of total soluble protein from MDEs. Our results indicated that: 1) pre-processing of bovine milk prior to storage does not affect the final exosome yield or the purity, 2) ExoQuick precipitation is better suited for MDE isolation than ultracentrifugation for bovine and human milk, and 3) TRIzol LS produced the highest RNA yield in bovine milk, whereas TRIzol LS, TRIzol+RNA Clean and Concentrator, and TRIzol LS+RNA Clean and Concentrator methods can be used for human milk. ### Competing Interest Statement The authors have declared no competing interest.