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

Posted: 2021-09-30 19:00:00
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.

参考サイト PubMed: exsome


6月 26, 2020 バイオアソシエイツ


2020年6月17日にNature Outlookでオンラインで公開された記事で、 イェール大学医学部のリウマチ学および臨床免疫学およびイェール大学医学部の元アレルギーおよび臨床免疫学のチーフであるPhilip Askenase教授(写真)は、「エクソソームはセンセーショナルな生物学的発見である」と述べている。膜に囲まれた小さな細胞内小胞は、研究されてきたすべての動物種のすべての細胞によって生産・分泌され、植物や細菌によっても放出される。…

ゲスト 925人 と メンバー 5人 がオンラインです