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Highly efficient and automated isolation technology for extracellular vesicles microRNA.

ABSTRACT: MicroRNA (miRNA) in extracellular vesicles (EVs) has great potential to be a promising marker in liquid biopsy. However, the present EV isolation methods, such as ultracentrifugation, have complicated and long-time operation, which impedes research on EV miRNA. The downstream complex miRNA extraction process will also significantly increase the detection cycle and loss. We first established a simple automated technique to efficiently extract target miRNAs in EVs from plasma based on Fe₃O₄@TiO₂ beads with high affinity and capture efficiency. We combined a heat-lysis method for quick and simple EV miRNA extraction and detection. The results indicated that our method has more RNA yield than TRIzol or a commercial kit and could complete EV enrichment and miRNA extraction in 30 min. Through the detection of miRNA-21, healthy people and lung cancer patients were distinguished, which verified the possibility of the application in clinical detection. The automated isolation technology for EV miRNA has good repeatability and high throughput, with great application potential in clinical diagnosis.

SUBMITTER: Di K

PROVIDER: S-EPMC9399425 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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Highly efficient and automated isolation technology for extracellular vesicles microRNA.

Di Kaili K Fan Boyue B Gu Xinrui X Huang Rongrong R Khan Adeel A Liu Chang C Shen Han H Li Zhiyang Z

Frontiers in bioengineering and biotechnology 20220810

MicroRNA (miRNA) in extracellular vesicles (EVs) has great potential to be a promising marker in liquid biopsy. However, the present EV isolation methods, such as ultracentrifugation, have complicated and long-time operation, which impedes research on EV miRNA. The downstream complex miRNA extraction process will also significantly increase the detection cycle and loss. We first established a simple automated technique to efficiently extract target miRNAs in EVs from plasma based on Fe<sub>3</su ...[more]

PMID: 36032725

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Project description:Extracellular vesicles (EVs) that circulate in body fluids possess significant potential for disease diagnosis. Their use in clinical settings, however, has been limited owing to lack of simple and robust isolation methods. To rectify this problem, a centrifugal device for automatic, fast, and efficient isolation of EVs from whole-blood, called Exodisc-B is presented in this paper. Methods: The device comprises a built-in chamber to facilitate plasma separation and two nanoporous filters-one for removing larger particles and the other for enriching EVs. The performance of the device in comparison to ultracentrifugation (UC) was evaluated by analyzing the yield, purity, protein and RNA content of the isolated EVs. Additionally, the EV protein marker expressions were measured by ELISA and statistically analyzed to differentiate prostate cancer patients from healthy donors. Results: Compared with the UC technique, the proposed device is capable of isolating at least an order of magnitude higher number of EVs with about 30-fold higher mRNA count within 40 min. Sandwich ELISA of EV-specific membrane proteins-CD9-CD81-confirmed that Exodisc-B can isolate EVs from a volume of whole blood as low as 30 µL with a capture efficiency exceeding 75%. The device also facilitates temporal monitoring of tumor progression within live mouse xenograft models over a period of 13 weeks while using minimal volumes of weekly collected blood samples. Further, in ELISA analyses of multiple cancer-related proteins, such as prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor 1 (EGFR1), and heat shock protein 90 (HSP90), extracted from EVs isolated from human plasma, 43 patients were differentiated from 30 healthy donors. Conclusion: The results demonstrated the ability of Exodisc-B to provide a rapid, sensitive, and point-of-care-type method for extracting intact EVs from small volumes of clinical blood samples for disease diagnosis and monitoring.

Dataset Information

Highly efficient and automated isolation technology for extracellular vesicles microRNA.

Publications

Highly efficient and automated isolation technology for extracellular vesicles microRNA.

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