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Mass spectrometric quantification of microRNAs in biological samples based on multistage signal amplification

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Abstract

This work describes a novel method for quantification of miRNAs based on multistage signal amplification (MSA) and liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The multistage signal amplification involves hybridization enrichment of miRNA targets with a DNA probe-magnetic bead conjugate, target recycling amplification with a duplex-specific nuclease, and acid hydrolysis of the reporter molecules producing free nucleobases. Nucleobases thus generated are quantified by LC-ESI-MS/MS with specificity and repeatability. Taking miR-21 as the model target, biological samples such as serum and cell cultures were analyzed by using the present protocol. The analytical results indicate that facile and cost-effective quantifications of miRNA targets can be achieved by using the popular LC-ESI-MS/MS technique, and very importantly, without an isolation of total RNAs from the sample prior to the quantitative assay. The assay for miR-21 detection had a linear calibration curve in the range from 0.2 pM to 0.25 nM with a limit of detection of 60 fM. Analysis of MCF-7 cells treated with toremifene (a potent inhibitor of breast cancer cell growth) revealed that the content of miRNA-21 decreased by ca. 50%, and the decrease was dose-dependent.

Graphical abstract: Mass spectrometric quantification of microRNAs in biological samples based on multistage signal amplification

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Supplementary files

Article information


Submitted
15 Oct 2019
Accepted
25 Dec 2019
First published
30 Dec 2019

Analyst, 2020, Advance Article
Article type
Paper

Mass spectrometric quantification of microRNAs in biological samples based on multistage signal amplification

X. Li, J. Zhao, R. Xu, L. Pan and Y. Liu, Analyst, 2020, Advance Article , DOI: 10.1039/C9AN02064K

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