Abstract

BackgroundExosomes are nano-sized extracellular vesicles containing different biomolecules such as proteins and microRNAs (miRNAs) that mediate intercellular communication. Recently, numerous studies have reported the important functions of exosomal miRNAs in disease development and the potential clinical application as diagnostic biomarkers. Up to now, the most commonly used methods to extract exosomes are ultracentrifugation (UC) and precipitation-based commercial kit (e.g., ExoQuick). Generally, both UC and ExoQuick method could co-isolate contaminating proteins along with exosomes, with the UC method yielding even purer exosomes than ExoQuick. However, the comparison of these two methods on co-precipitated free miRNAs is still unknown.MethodsIn this study, we isolated exosomes from the human serum with exogenously added cel-miR-39 by UC and ExoQuick and compared the proportion of cel-miR-39 co-precipitated with exosomes extracted by these two methods.ResultsUsing exogenous cel-miR-39 as free miRNAs in serum, we concluded that ExoQuick co-isolates a small proportion of free miRNAs while UC hardly precipitates any free miRNAs. We also found that incubation at 37 °C for 1 h could decrease the proportion of free miRNAs, and exosomal miRNAs like miR-126 and miR-152 also decreased when RNase A was used. In conclusion, our findings provide essential information about the details of serum exosome isolation methods for further research on exosomal miRNAs.

Highlights

  • Exosomes are small secreted extracellular vesicles of 30–200 nm in diameter with the same structure as cell membrane [1,2,3]

  • We isolated exosomes from the human serum with exogenously added cel-miR-39 by UC and ExoQuick and compared the proportion of cel-miR-39 co-precipitated with exosomes extracted by these two methods

  • Using exogenous cel-miR-39 as free miRNAs in serum, we concluded that ExoQuick co-isolates a small proportion of free miRNAs while UC hardly precipitates any free miRNAs

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Summary

Introduction

Exosomes are small secreted extracellular vesicles of 30–200 nm in diameter with the same structure as cell membrane [1,2,3] They convey intercellular communications by delivery of biomolecules and affect multiple physiological processes under normal or diseased conditions [4,5,6]. They originate from multivesicular bodies (MVBs) which contain many small vesicles called intraluminal endosomal vesicles (ILVs) and the ILVs become exosomes when the MVBs fuse with the plasma membrane, releasing the ILVs into the extracellular space [7]. The comparison of these two methods on coprecipitated free miRNAs is still unknown

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