Tags

Type your tag names separated by a space and hit enter

Two-step magnetic bead-based (2MBB) techniques for immunocapture of extracellular vesicles and quantification of microRNAs for cardiovascular diseases: A pilot study.
PLoS One. 2020; 15(2):e0229610.Plos

Abstract

Extracellular vesicles (EVs) have attracted increasing attention because of their potential roles in various biological processes and medical applications. However, isolation of EVs is technically challenging mainly due to their small and heterogeneous size and contaminants that are often co-isolated. We have thus designed a two-step magnetic bead-based (2MBB) method for isolation a subset of EVs as well as their microRNAs from samples of a limited amount. The process involves utilizing magnetic beads coated with capture molecules that recognize EV surface markers, such as CD63. Captured EVs could be eluted from beads or lyzed directly for subsequent analysis. In this study, we used a second set of magnetic beads coated with complementary oligonucleotides to isolate EV-associated microRNAs (EV-miRNAs). The efficiencies of 2MBB processes were assessed by reverse transcription-polymerase chain reaction (RT-PCR) with spiked-in exogenous cel-miR-238 molecules. Experimental results demonstrated the high efficiency in EV enrichment (74 ± 7%, n = 4) and miRNA extraction (91 ± 4%, n = 4). Transmission electron micrographs (TEM) and nanoparticle tracking analysis (NTA) show that captured EVs enriched by 2MBB method could be released and achieved a higher purity than the differential ultracentrifugation (DUC) method (p < 0.001, n = 3). As a pilot study, EV-miR126-3p and total circulating cell-free miR126-3p (cf-miR126-3p) in eight clinical plasma samples were measured and compared with the level of protein markers. Compared to cf-miR126-3p, a significant increase in correlations between EV-miR126-3p and cardiac troponin I (cTnI) and N-terminal propeptide of B-type natriuretic peptide (NT-proBNP) was detected. Furthermore, EV-miR126-3p levels in plasma samples from healthy volunteers (n = 18) and high-risk cardiovascular disease (CVD) patients (n = 10) were significantly different (p = 0.006), suggesting EV-miR126 may be a potential biomarker for cardiovascular diseases. 2MBB technique is easy, versatile, and provides an efficient means for enriching EVs and EV-associated nucleic acid molecules.

Links

PMC Free PDF

Authors+Show Affiliations

Chen S
Institution of NanoEngineering and MicroSystems, National Tsing Hua University, Hsinchu, Taiwan.
Shiesh SC
Department of Medical Laboratory Science and Biotechnology, National Cheng Kung University, Tainan City, Taiwan.
Lee GB
Institution of NanoEngineering and MicroSystems, National Tsing Hua University, Hsinchu, Taiwan. Department of Power Mechanical Engineering, National Tsing Hua University, Hsinchu, Taiwan. Institution of Biomedical Engineering, National Tsing Hua University, Hsinchu, Taiwan.
Chen C
Institution of NanoEngineering and MicroSystems, National Tsing Hua University, Hsinchu, Taiwan. Department of Power Mechanical Engineering, National Tsing Hua University, Hsinchu, Taiwan.

MeSH

BiomarkersCardiovascular DiseasesExtracellular VesiclesHumansImmunomagnetic SeparationMagnetic PhenomenaMicroRNAsPilot ProjectsROC Curve

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

32101583

Citation

Chen, Shi, et al. "Two-step Magnetic Bead-based (2MBB) Techniques for Immunocapture of Extracellular Vesicles and Quantification of microRNAs for Cardiovascular Diseases: a Pilot Study." PloS One, vol. 15, no. 2, 2020, pp. e0229610.
Chen S, Shiesh SC, Lee GB, et al. Two-step magnetic bead-based (2MBB) techniques for immunocapture of extracellular vesicles and quantification of microRNAs for cardiovascular diseases: A pilot study. PLoS One. 2020;15(2):e0229610.
Chen, S., Shiesh, S. C., Lee, G. B., & Chen, C. (2020). Two-step magnetic bead-based (2MBB) techniques for immunocapture of extracellular vesicles and quantification of microRNAs for cardiovascular diseases: A pilot study. PloS One, 15(2), e0229610. https://doi.org/10.1371/journal.pone.0229610
Chen S, et al. Two-step Magnetic Bead-based (2MBB) Techniques for Immunocapture of Extracellular Vesicles and Quantification of microRNAs for Cardiovascular Diseases: a Pilot Study. PLoS One. 2020;15(2):e0229610. PubMed PMID: 32101583.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Two-step magnetic bead-based (2MBB) techniques for immunocapture of extracellular vesicles and quantification of microRNAs for cardiovascular diseases: A pilot study. AU - Chen,Shi, AU - Shiesh,Shu-Chu, AU - Lee,Gwo-Bin, AU - Chen,Chihchen, Y1 - 2020/02/26/ PY - 2019/09/23/received PY - 2020/02/10/accepted PY - 2020/2/27/entrez PY - 2020/2/27/pubmed PY - 2020/6/12/medline SP - e0229610 EP - e0229610 JF - PloS one JO - PLoS One VL - 15 IS - 2 N2 - Extracellular vesicles (EVs) have attracted increasing attention because of their potential roles in various biological processes and medical applications. However, isolation of EVs is technically challenging mainly due to their small and heterogeneous size and contaminants that are often co-isolated. We have thus designed a two-step magnetic bead-based (2MBB) method for isolation a subset of EVs as well as their microRNAs from samples of a limited amount. The process involves utilizing magnetic beads coated with capture molecules that recognize EV surface markers, such as CD63. Captured EVs could be eluted from beads or lyzed directly for subsequent analysis. In this study, we used a second set of magnetic beads coated with complementary oligonucleotides to isolate EV-associated microRNAs (EV-miRNAs). The efficiencies of 2MBB processes were assessed by reverse transcription-polymerase chain reaction (RT-PCR) with spiked-in exogenous cel-miR-238 molecules. Experimental results demonstrated the high efficiency in EV enrichment (74 ± 7%, n = 4) and miRNA extraction (91 ± 4%, n = 4). Transmission electron micrographs (TEM) and nanoparticle tracking analysis (NTA) show that captured EVs enriched by 2MBB method could be released and achieved a higher purity than the differential ultracentrifugation (DUC) method (p < 0.001, n = 3). As a pilot study, EV-miR126-3p and total circulating cell-free miR126-3p (cf-miR126-3p) in eight clinical plasma samples were measured and compared with the level of protein markers. Compared to cf-miR126-3p, a significant increase in correlations between EV-miR126-3p and cardiac troponin I (cTnI) and N-terminal propeptide of B-type natriuretic peptide (NT-proBNP) was detected. Furthermore, EV-miR126-3p levels in plasma samples from healthy volunteers (n = 18) and high-risk cardiovascular disease (CVD) patients (n = 10) were significantly different (p = 0.006), suggesting EV-miR126 may be a potential biomarker for cardiovascular diseases. 2MBB technique is easy, versatile, and provides an efficient means for enriching EVs and EV-associated nucleic acid molecules. SN - 1932-6203 UR - https://www.unboundmedicine.com/medline/citation/32101583/Two_step_magnetic_bead_based__2MBB__techniques_for_immunocapture_of_extracellular_vesicles_and_quantification_of_microRNAs_for_cardiovascular_diseases:_A_pilot_study_ DB - PRIME DP - Unbound Medicine ER -