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Methods for Collection of Extracellular Vesicles and Their Content RNA as Liquid Biopsy for Lung Cancer Detection: Application of Differential Centrifugation and Annexin A5 Coated Beads

Authors :
Mei-Chia Wang
Guan-Yu Gong
Chih-Liang Wang
How-Wen Ko
Rong-Xuan Weng
Pi-Yueh Chang
Chiuan-Chian Chiou
Source :
Current Issues in Molecular Biology, Vol 44, Iss 5, Pp 2374-2386 (2022)
Publication Year :
2022
Publisher :
MDPI AG, 2022.

Abstract

Extracellular vesicles (EVs) contain abundant extracellular RNA (exRNA), which can be a valuable source of liquid biopsy. However, as various RNA species exist in different types of EVs, lack of detailed characterization of these RNA species and efficient collection methods limits the clinical application of exRNA. In the present study, we measured two mRNAs, CK19 and PCTK1; one lncRNA, MALAT1; and two miRNAs, miR21 and miR155, in different EV fractions separated by differential centrifugation or captured by magnetic beads coated with annexin A5 (ANX beads). The results showed that in a cultured medium, the majority of mRNA and lncRNA exist in larger EVs, whereas miRNA exist in both large and small EVs from the differential centrifugation fractions. All these RNA species exist in ANX beads captured EVs. We then used ANX beads to capture EVs in plasma samples from non-small-cell lung cancer patients and age-matched healthy volunteers. We found that the ANX bead capturing could efficiently improve RNA detection from human plasma, compared with direct extraction of RNA from plasma. Using ANX-bead capturing and reverse transcription and quantitative PCR, we detected significantly higher levels of CK19 mRNA, MALAT1 lncRNA, and miR155 miRNA in the plasma of lung cancer patients. These facts suggested the collection methods strongly affect the results of exRNA measurement from EVs, and that ANX beads can be a useful tool for detecting exRNA from plasma samples in clinical application.

Details

Language :
English
ISSN :
14673045 and 14673037
Volume :
44
Issue :
5
Database :
Directory of Open Access Journals
Journal :
Current Issues in Molecular Biology
Publication Type :
Academic Journal
Accession number :
edsdoj.f3d53c2672954153a52397b985b9797e
Document Type :
article
Full Text :
https://doi.org/10.3390/cimb44050162