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Issue 13, 2018
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Naked-eye and electrochemical detection of isothermally amplified HOTAIR long non-coding RNA

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Abstract

An inexpensive, simple and rapid sensor platform capable of detecting cancer-related long non-coding RNA (lncRNA) with high accuracy is of great interest in the field of molecular diagnostics. Herein, we report on the development of a new colorimetric and electrochemical assay platform for long non-coding HOX transcript antisense intergenic RNA (HOTAIR) detection. Isothermal reverse transcription-recombinase polymerase amplification (RT-RPA) was performed to amplify HOTAIR sequences from a RNA pool extracted from a designated number of ovarian cancer cells and a small cohort of plasma samples derived from patients with ovarian cancer. During RT-RPA, biotinylated dUTPs were randomly incorporated in the amplified product. Subsequently, HOTAIR amplicons were magnetically purified and isolated followed by a horseradish peroxidase (HRP)-catalyzed colorimetric reaction in the presence of the 3,3′,5,5′-tetramethylbenzidine (TMB)/H2O2 system. We finally introduced three potential readout methods for HOTAIR detection – (i) naked-eye visualisation of the color change for a quick screening of the target, (ii) quantitative absorbance measurement by UV-vis, and (iii) amperometric quantification using the electrochemical properties of TMB. The assay has shown excellent reproducibility (% RSD = <5%, for n = 3) and sensitivity (10 cells/ per mL) while detecting HOTAIR in cancer cell lines and patient samples. The expression of HOTAIR in clinical samples was also verified with a standard RT-qPCR method. We believe that our proof of concept assay may find potential relevance for the routine clinical screening of cancer-associated lncRNAs.

Graphical abstract: Naked-eye and electrochemical detection of isothermally amplified HOTAIR long non-coding RNA

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Supplementary files

Article information


Submitted
31 Dec 2017
Accepted
15 Mar 2018
First published
23 Mar 2018

Analyst, 2018,143, 3021-3028
Article type
Paper

Naked-eye and electrochemical detection of isothermally amplified HOTAIR long non-coding RNA

Md. N. Islam, S. Moriam, M. Umer, H. Phan, C. Salomon, R. Kline, N. Nguyen and M. J. A. Shiddiky, Analyst, 2018, 143, 3021
DOI: 10.1039/C7AN02109G

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