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

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, stora...

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Autores principales:	Sanoji Wijenayake, Shafinaz Eisha, Zoya Tawhidi, Michael A Pitino, Michael A Steele, Alison S Fleming, Patrick O McGowan
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/ad57b43f7fdb4831bb42a51af8d65046
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spelling	oai:doaj.org-article:ad57b43f7fdb4831bb42a51af8d650462021-12-02T20:13:56ZComparison of methods for pre-processing, exosome isolation, and RNA extraction in unpasteurized bovine and human milk.1932-620310.1371/journal.pone.0257633https://doaj.org/article/ad57b43f7fdb4831bb42a51af8d650462021-01-01T00:00:00Zhttps://doi.org/10.1371/journal.pone.0257633https://doaj.org/toc/1932-6203Milk 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.Sanoji WijenayakeShafinaz EishaZoya TawhidiMichael A PitinoMichael A SteeleAlison S FlemingPatrick O McGowanPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 16, Iss 9, p e0257633 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Sanoji Wijenayake Shafinaz Eisha Zoya Tawhidi Michael A Pitino Michael A Steele Alison S Fleming Patrick O McGowan Comparison of methods for pre-processing, exosome isolation, and RNA extraction in unpasteurized bovine and human milk.
description	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.
format	article
author	Sanoji Wijenayake Shafinaz Eisha Zoya Tawhidi Michael A Pitino Michael A Steele Alison S Fleming Patrick O McGowan
author_facet	Sanoji Wijenayake Shafinaz Eisha Zoya Tawhidi Michael A Pitino Michael A Steele Alison S Fleming Patrick O McGowan
author_sort	Sanoji Wijenayake
title	Comparison of methods for pre-processing, exosome isolation, and RNA extraction in unpasteurized bovine and human milk.
title_short	Comparison of methods for pre-processing, exosome isolation, and RNA extraction in unpasteurized bovine and human milk.
title_full	Comparison of methods for pre-processing, exosome isolation, and RNA extraction in unpasteurized bovine and human milk.
title_fullStr	Comparison of methods for pre-processing, exosome isolation, and RNA extraction in unpasteurized bovine and human milk.
title_full_unstemmed	Comparison of methods for pre-processing, exosome isolation, and RNA extraction in unpasteurized bovine and human milk.
title_sort	comparison of methods for pre-processing, exosome isolation, and rna extraction in unpasteurized bovine and human milk.
publisher	Public Library of Science (PLoS)
publishDate	2021
url	https://doaj.org/article/ad57b43f7fdb4831bb42a51af8d65046
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Comparison of methods for pre-processing, exosome isolation, and RNA extraction in unpasteurized bovine and human milk.

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