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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

差速离心 核糖核酸 离心 液体活检 RNA提取 小RNA 分子生物学 胞外囊泡 肺癌 信使核糖核酸 化学 生物 色谱法 微泡 癌症 病理 基因 生物化学 医学 遗传学
作者
Mei-Chia Wang,Guanyu Gong,Chih‐Liang Wang,How‐Wen Ko,Rong-Xuan Weng,Pi‐Yueh Chang,Chiuan-Chian Chiou
出处
期刊:Current Issues in Molecular Biology [MDPI AG]
卷期号:44 (5): 2374-2386 被引量:5
标识
DOI:10.3390/cimb44050162
摘要

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.
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