Isolation of selected miRNAs in extracellular vesicles using two magnetic-bead-based enrichments

• Main Authors: Lin, Hsing-Yu, 林星佑
• Other Authors: Chen, Chih-Chen
• Format: Others
• Language: zh-TW
• Published: 2017
• Online Access: http://ndltd.ncl.edu.tw/handle/3ecbkd

碩士 === 國立清華大學 === 動力機械工程學系 === 106 === The method of isolateing plasma extracellular vesicles and extacting EV-contained microRNA has been investigated systematically, which performance is assessed by using real-time PCR quantififcation. Magnetic beads are utililzed for the development of a simple EV isolating technique that is amiable for microfluidic integration. Magnetic beads coated with CD63 antibodies are mixed with samples to isolate CD63+ EVs. EV-contained miR-21, miR-126 and miR-208a, as biomarkers for cardiovascular diseases, are extracted subsequently after mixing EV lysate with magnetic beads coated with complementary miRNA sequences. ~ 70% EVs are isolated from conditioned cell culture medium after 18 h of mixing with magnetic beads, which is assessed with a Bioanalyzer as well as nanoparticle tracking analysis. The specificity of microRNA-bead isolation is verified with sequencing results. Furthermore, calibration curves of RT-qPCR results have been established using synthesized microRNA molecules. Preliminary data indicate 1.3 and 14811 fM of miR-21 and miR-126 are present in healthy human plasma CD63+ EVs. Finally, an automated microfluidic device has shown comparable CD63+ EV isolation results. In summary, this research has demonstrated that the proposed method is capable of isolating EV-contained microRNA in human plasma samples and may potentially be implemented on microfluidic chips.