Comparison of isolation methods of exosomes and exosomal RNA from cell culture medium and serum

Exosomes are cell-derived vesicles and are abundant in biological fluids; they contain RNA molecules which may serve as potential diagnostic biomarkers in 'precision medicine'. To promote the clinical application of exosomal RNA (exoRNA), many isolation methods must be compared and validat...

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Vydané v:International journal of molecular medicine Ročník 40; číslo 3; s. 834 - 844
Hlavní autori: Tang, Yue-Ting, Huang, Yi-Yao, Zheng, Lei, Qin, Si-Hua, Xu, Xu-Ping, An, Tai-Xue, Xu, Yong, Wu, Ying-Song, Hu, Xiu-Mei, Ping, Bao-Hong, Wang, Qian
Médium: Journal Article
Jazyk:English
Vydavateľské údaje: Greece D.A. Spandidos 01.09.2017
Spandidos Publications
Spandidos Publications UK Ltd
Predmet:
A549 Cells
Cell culture
cell culture medium
Culture Media, Conditioned - chemistry
Cytological research
Efficiency
exosomal RNA
exosomes
Exosomes - chemistry
Humans
Innovations
isolation methods
Laboratories
Methods
Organelles
Phenols
Properties
Proteins
RNA - biosynthesis
RNA - chemistry
RNA - isolation & purification
serum
United States > US
ISSN:1107-3756, 1791-244X, 1791-244X
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Shrnutí:Exosomes are cell-derived vesicles and are abundant in biological fluids; they contain RNA molecules which may serve as potential diagnostic biomarkers in 'precision medicine'. To promote the clinical application of exosomal RNA (exoRNA), many isolation methods must be compared and validated. Exosomes in cell culture medium (CCM) and serum may be isolated using ultracentrifugation (UC), ExoQuick or Total Exosome Isolation Reagent (TEI), and exoRNA may be extracted using TRIzol-LS, SeraMir, Total Exosome RNA Isolation (TER), HiPure Liquid RNA/miRNA kit (HLR), miRNeasy or exoRNeasy. ExoRNA was assessed using NanoDrop, Bioanalyzer 2100, quantitative polymerase chain reaction and high-throughput sequencing. UC showed the lowest recovery of particles, but the highest protein purity for exosome isolation. For isolation of exoRNA, we found that combinations of the TEI and TER methods resulted in high extraction efficiency and purity of small RNA obtained using CCM. High yield and a narrow size distribution pattern of small RNA were shown in exoRNA isolated by exoRNeasy from serum. In RNA profile analysis, the small RNA constituent ratio, miRNA content and amount varied as a result of methodological differences. This study showed that different methods may introduce variations in the concentration, purity and size of exosomes and exoRNA. Herein we discuss the advantages and disadvantages of each method and their application to different materials, therefore providing a reference according to research design.
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Contributed equally
ISSN:1107-3756
1791-244X
1791-244X
DOI:10.3892/ijmm.2017.3080