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

Abstract

Milk is a highly complex, heterogeneous biological fluid that contains non-nutritive, bioactive 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, storage, and exosome isolation. In this study, we tested: 1) three pre-processing methods to remove cream, fat, cellular debris, and casein proteins from bovine milk to determine whether pre-processing of whole milk prior to long-term storage improves MDE isolations, 2) the suitability of two standard exosome isolation methods for MDE fractionation, and 3) four extraction protocols for obtaining high quality RNA from bovine and human MDEs. MDEs were characterized via Transmission Electron Microscopy (TEM), Nanoparticle Tracking Analysis (NTA), and western immunoblotting for CD9, CD63, and Calnexin protein markers. We also present an optimized method of TEM sample preparation for MDEs. Our results indicate that: 1) Removal of cream and fat globules from unpasteurized bovine milk, prior to long-term storage, improves the MDE yield but not purity, 2) Differential ultracentrifugation (DUC) combined with serial filtration is better suited for bovine MDE isolation compared to ExoQuick (EQ) combined with serial filtration, however both methods were comparable for human milk, and 3) TRIzol LS is better suited for RNA extraction from bovine MDEs isolated by EQ and DUC methods. 4) TRIzol LS, TRIzol+RNA Clean and Concentrator, and TRIzol LS+RNA Clean and Concentrator methods can be used for RNA extractions from human MDEs isolated by EQ, yet the TRIzol LS method is better suited for human MDEs isolated by DUC. The QIAzol + miRNeasy Mini Kit produced the lowest RNA yield for bovine and human MDEs.

Putative Internal Control Genes in Bovine Milk Small Extracellular Vesicles Suitable for Normalization in Quantitative Real Time-Polymerase Chain Reaction

“…There is no easy solution to the question of which algorithm should be used to identify the best putative internal control genes regardless of sample variations [28,29]. It might also be considered that RNA stability and putative internal control genes expression stability may be changed or influenced by the sample collection, pre-processing procedure, method of RNA extraction, sample storage, and time [30,31]. Moreover, using artificial putative internal control genes such as 18S rRNA and 28S rRNA in milk sEVs for normalization in qRT-PCR is a questionable approach.…”

Assessment of microRNA profiles in small extracellular vesicles isolated from bovine colostrum with different immunoglobulin G concentrations

“…The RNA yield was highest (45.46 ng/μL) using miRCURY Exosome Cell/Urine/ CSF method for sEV purification followed by miRNeasy Mini kits (Table 1). The RNA concentrations in bovine colostrum exosome was reported to be ranging from 0 to 100 ng/μL using different kits as reported by Wijenayake et al (2021). This combination was subsequently used for extracting sEV from 8 bovine colostrum samples with different IgG concentrations.…”

The Therapeutic Potential of Milk Extracellular Vesicles on Colorectal Cancer

“…Exosomes are now isolated and purified from cell cultures and bodily fluids using different methods [ 35 ]. However, the separated exosomes’ pureness varies, which is probably related to the contaminated particles, different sub-types of EVs, sample viscosity, and proteins of milk [ 36 ]. Furthermore, poor exosomal purity is because most existing separation methods fail to fully isolate exosomes from compounds that have identical biophysical characteristics, such as lipoproteins [ 35 ].…”

“…As a result, they may take part in protein expression and signaling cascades between cells [ 75 , 134 ]. In addition, these elements play an important part in the immune system’s development, inflammatory regulation, and cell proliferation and progression [ 36 , 48 ]. The exosomal mRNA of mice can be translated into proteins in human cells, for example, when human cells are treated with mouse exosomes [ 135 ].…”