http://dx.doi.org/10.1021/ac101133x">
 

Document Type

Journal Article

Department/Unit

Department of Chemistry

Title

Direct quantification of single-molecules of microRNA by total internal reflection fluorescence microscopy

Language

English

Abstract

MicroRNAs (miRNAs) express differently in normal and cancerous tissues and thus are regarded as potent cancer biomarkers for early diagnosis. However, the short length and low abundance of miRNAs have brought challenges to the established detection assay in terms of sensitivity and selectivity. In this work, we present a novel miRNA detection assay in single-molecule level with total internal reflection fluorescence microscopy (TIRFM). It is a solution-based hybridization detection system that does not require pretreatment steps such as sample enrichment or signal amplification. The hsa-miR-21 (miR-21) is chosen as target miRNA for its significant elevated content in a variety of cancers as reported previously. Herein, probes of complementary single-stranded oligonucleotide were hybridized in solution to miR-21 and labeled with fluorescent dye YOYO-1. The fluorescent hybrids were imaged by an electron-multiplying charge-coupled device (EMCCD) coupled TIRFM system and quantified by single-molecule counting. This single molecule detection (SMD) assay shows a good correlation between the number of molecules detected and the factual concentration of miRNA. The detection assay is applied to quantify the miR-21 in extracted total RNA samples of cancerous MCF-7 cells, HepG2 cells, and normal HUVEC cells, respectively. The results agreed very well with those from the prevalent real-time polymerase chain reaction (qRT-PCR) analysis. This assay is of high potential for applications in miRNA expression profiling and early cancer diagnosis. © 2010 American Chemical Society.

Publication Date

2010

Source Publication Title

Analytical Chemistry

Volume

82

Issue

16

Start Page

6911

End Page

6918

Publisher

American Chemical Society

ISSN (print)

00032700

ISSN (electronic)

15206882

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