DNA in serum extracellular vesicles is stable under different storage conditions.

Yang Jin,Youliang Wang,Jianming Xu,Gairong Zhang,Zongying Wang,Cong Cui,Yan Wang,Keyan Chen,Yong Shao,Chuanhua Zhao,Lijuan Du,Ru Jia,Rongrui Liu,Jianzhi Liu,Lihua Gao,Yuling Chen,Xianwen Hu,Li Lin,Hongbo Yin ,Lie-Jun Liu ,Zhao-Li Tan

doi:10.1186/s12885-016-2783-2

Yang Jin, Youliang Wang + Show 19 more

Open Access

https://doi.org/10.1186/s12885-016-2783-2

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Abstract

BackgroundExtracellular vesicles (EVs), including exosomes, microvesicles, and apoptotic bodies, can be secreted by most cell types and released in perhaps all biological fluids. EVs contain multiple proteins, specific lipids and several kinds of nucleic acids such as RNAs and DNAs. Studies have found that EVs contain double-stranded DNA and that genetic information has a certain degree of consistency with tumor DNA. Therefore, if genes that exist in exosomes are stable, we may be able to use EVs genetic testing as a new means to monitor gene mutation.MethodsIn this study, EVs were extracted from serum under various storage conditions (4 °C, room temperature and repeated freeze-thaw). We used western blotting to examine the stability of serum EVs. Then, we extracted DNA from EVs and tested the concentration changing under different conditions. We further assessed the stability of EVs DNA s using polymerase chain reaction (PCR) and Sanger sequencing.ResultsEVs is stable under the conditions of 4 °C (for 24 h, 72 h, 168 h), room temperature (for 6 h, 12 h, 24 h, 48 h) and repeated freeze-thaw (after one time, three times, five times). Also, serum DNA is mainly present in EVs, especially in exosomes, and that the content and function of DNA in EVs is stable whether in a changing environment or not. We showed that EVs DNA stayed stable for 1 week at 4 °C, 1 day at room temperature and after repeated freeze-thaw cycles (less than three times). However, DNA from serum EVs after 2 days at room temperature or after five repeated freeze-thaw cycles could be used for PCR and sequencing.ConclusionsSerum EVs and EVs DNA can remain stable under different environments, which is the premise that EVs could serve as a novel means for genetic tumor detection and potential biomarkers for cancer diagnostics and prognostics.

Highlights

Extracellular vesicles (EVs), including exosomes, microvesicles, and apoptotic bodies, can be secreted by most cell types and released in perhaps all biological fluids
Exosomes are nanometer-sized EVs of endocytic origin that are secreted by most cell types, under physiological and pathological conditions upon fusion of multivesicular bodies (MVBs) with the plasma membrane (PM) [2,3,4,5]
Compared with different types of RNA, such as messenger RNA, microRNA, and noncoding RNA, which have been shown in the Exocarta database [19], less is known about EVs DNA content, some types of DNA have been reported, such as single-stranded DNA [22], mitochondrial DNA [23] and plasmid DNA [24]

Summary

Introduction

Extracellular vesicles (EVs), including exosomes, microvesicles, and apoptotic bodies, can be secreted by most cell types and released in perhaps all biological fluids. Extracellular vesicles (EVs) are cell-derived membrane vesicles, including exosomes, microvesicles, and other types of membrane vesicles [1]. Exosomes are nanometer-sized EVs of endocytic origin that are secreted by most cell types, under physiological and pathological conditions upon fusion of multivesicular bodies (MVBs) with the plasma membrane (PM) [2,3,4,5]. The question remains as to whether the DNA cargo is randomly sorted or if it is systematically packed into EVs. A recent report by Mark et al found that exosomes contain double-stranded DNA (dsDNA)and that exosome DNA can serve as a novel biomarker for cancer detection [25]. An increasing number of studies have emerged showing that different types of DNA in EVs may be associated with various biological functions [26,27,28]

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