Indirect and sensitive determination of microRNAs by magnetic field-assisted capillary sieving electrophoresis combined with catalytic hairpin assembly

IF 2.8 3区 工程技术 Q2 CHEMISTRY, ANALYTICAL
Jinfeng Ning, Junlan Ye, Qingqing Wang, Wei Wang
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Abstract

To determine multiple microRNAs (miRNAs) from cells simultaneously is essential for understanding biological functions. Capillary electrophoresis (CE) can simultaneously determine multiple miRNAs by separation. Nevertheless, similar lengths and low concentrations in cells make miRNAs hard to separate and detect. In this study, CE with laser-induced fluorescence detection was combined with catalytic hairpin assembly (CHA) to determine three miRNAs, miR-21, miR-31, and miR-122. The amplification products of CHA, which were DNA duplexes, were designed to have different lengths for different miRNAs. This allowed for easy separation of the duplexes of different miRNAs by CE. The indirect determination of miRNAs was then achieved by separating and detecting these duplexes. A magnetic field was first applied on the capillary sieving electrophoresis to assist in the separation of the duplexes. Under the optimal conditions, the three duplexes could be completely separated within 2.5 min with the detection limits of miRNAs in the range 1.12–4.05 × 10−15 M. MiR-21 and miR-31 were successfully determined from Hela cells, while miR-122 was determined from chicken livers by this method. The recoveries ranged from 97.5% to 118%. The developed method was sensitive and reliable for miRNA determination.

利用磁场辅助毛细管筛分电泳结合催化发夹组装技术间接灵敏地测定 microRNA。
同时测定细胞中的多种微RNA(miRNA)对于了解生物功能至关重要。毛细管电泳(CE)可通过分离同时测定多种 miRNA。然而,由于长度相似且在细胞中浓度较低,miRNA 难以分离和检测。本研究将激光诱导荧光检测的毛细管电泳与催化发夹组装(CHA)相结合,测定了三种 miRNA:miR-21、miR-31 和 miR-122。CHA的扩增产物是DNA双链体,针对不同的miRNA设计了不同的长度。这样就可以用 CE 法轻松分离不同 miRNA 的双链体。然后通过分离和检测这些双链体来间接测定 miRNA。首先在毛细管筛分电泳上施加磁场,以帮助分离双链体。在最佳条件下,三个双链体可在 2.5 分钟内完全分离,miRNA 的检测限在 1.12-4.05 × 10-15 M 之间。回收率为 97.5% 至 118%。该方法灵敏可靠,适用于 miRNA 的测定。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Journal of separation science
Journal of separation science 化学-分析化学
CiteScore
6.30
自引率
16.10%
发文量
408
审稿时长
1.8 months
期刊介绍: The Journal of Separation Science (JSS) is the most comprehensive source in separation science, since it covers all areas of chromatographic and electrophoretic separation methods in theory and practice, both in the analytical and in the preparative mode, solid phase extraction, sample preparation, and related techniques. Manuscripts on methodological or instrumental developments, including detection aspects, in particular mass spectrometry, as well as on innovative applications will also be published. Manuscripts on hyphenation, automation, and miniaturization are particularly welcome. Pre- and post-separation facets of a total analysis may be covered as well as the underlying logic of the development or application of a method.
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