Naked-eye and electrochemical detection of isothermally amplified HOTAIR long non-coding RNA. Issue 13 (18th April 2018)

• Record Type: Journal Article
• Authors: Islam, Md. Nazmul; Moriam, Sofia; Umer, Muhammad; Phan, Hoang-Phuong; Salomon, Carlos; Kline, Richard; Nguyen, Nam-Trung; Shiddiky, Muhammad J. A.
• Is Part Of: Analyst. Volume 143:Issue 13(2018)
• Page Start: 3021
• Page End: 3028
• Publication Date: 2018-04-18
• Subjects: Chemistry, Analytic -- Periodicals; 543
• DOI: 10.1039/c7an02109g
• Languages: English
• ISSNs: 0003-2654

Abstract

Abstract : A naked-eye, colorimetric and electrochemical detection of HOTAIR long non-coding RNA has been demonstrated. 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)/H2 O2 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.