Evaluation of Small Extracellular Vesicles Isolation Methods from Human Serum for Downstream miRNA Profiling

Yong Ling  Sou; Paulina Pei Suu  Tan; Nur Afrina  Muhamad Hendiri; Wickneswari  Ratnam; Tilakavati  Karupaiah; William M Chilian; Shamsul Mohd  Zain; Sharifah Zamiah  Syed Abdul Kadir; Yan  Pan; Yuh Fen Pung

doi:10.55230/mabjournal.v54i1.3357

Authors

Yong Ling Sou Division of Biomedical Science, Faculty of Science and Engineering, University of Nottingham Malaysia, 43500 Selangor, Malaysia
Paulina Pei Suu Tan Division of Biomedical Science, Faculty of Science and Engineering, University of Nottingham Malaysia, 43500 Selangor, Malaysia
Nur Afrina Muhamad Hendiri Department of Electron Microscopy, Institute of Medical Research, National Institutes of Health, Ministry of Health Malaysia, 40170 Selangor, Malaysia
Wickneswari Ratnam Department of Biological Sciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, Bangi 43600, Malaysia
Tilakavati Karupaiah School of Biosciences, Faculty of Health and Medical Sciences, Taylor’s University Lakeside Campus, 47500 Selangor, Malaysia
William M Chilian Department of Integrative Medical Sciences, Northeast Ohio Medical University, 44272 Ohio, United States
Shamsul Mohd Zain Department of Pharmacology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia
Sharifah Zamiah Syed Abdul Kadir Department of Pharmacology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia
Yan Pan Division of Biomedical Science, Faculty of Science and Engineering, University of Nottingham Malaysia, 43500 Selangor, Malaysia
Yuh Fen Pung
YuhFen.Pung@nottingham.edu.my
Division of Biomedical Science, Faculty of Science and Engineering, University of Nottingham Malaysia, 43500 Selangor, Malaysia https://orcid.org/0000-0001-6195-3970

Keywords:

Commercial kits, exosome, isolation, polyethylene glycol, purification, ultracentrifugation

Abstract

Exosomes are a type of extracellular vesicles that carry distinct profiles of biomolecules such as lipids, proteins, DNAs, and RNAs. Despite many years of research, there is still a lack in standardized method to isolate exosomes from clinical samples for their downstream applications. Thus, this study compared three different methods, which are the differential ultracentrifugation (DUC), polyethylene glycol-based precipitation (PEG), and a combination of both (PEG+UC) to isolate exosomes from human serum. The isolated exosomes were evaluated by their size distribution, recovered particle concentration, particle to protein ratio, exosomal marker expression, and miRNA recovery. Our results indicated that all three methods successfully isolated exosomes, however, with varying yield and purity. In particular, PEG+UC produced exosomes of both high yield and high purity, DUC produced exosomes of both low yield and low purity, whereas PEG produced exosomes of high yield but low purity. Using miR-30d-5p and let-7i-5p as selected targets, our qPCR results indicated significant differences in terms of exosomal miRNA recovery between all three methods. Overall, the PEG+UC method appeared to be a less labour-intensive alternative that can isolate exosomes of both high yield and high purity from human serum without compromising the yield of miRNAs.

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