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

Islam, Md. Nazmul; Moriam, Sofia; Umer, Muhammad; Phan, Hoang-Phuong; Salomon, Carlos; Kline, Richard; Nguyen, Nam-Trung; Shiddiky, Muhammad J.A.

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.

Citation

Islam, M. N., Moriam, S., Umer, M., Phan, H.-P., Salomon, C., Kline, R., Nguyen, N.-T., & Shiddiky, M. J. (2018). Naked-eye and electrochemical detection of isothermally amplified HOTAIR long non-coding RNA. Analyst, https://doi.org/10.1039/c7an02109g

• Journal Article Type: Article
• Acceptance Date: Mar 15, 2018
• Online Publication Date: Mar 23, 2018
• Publication Date: Mar 23, 2018
• Deposit Date: Jun 25, 2025
• Journal: Analyst
• Print ISSN: 0003-2654
• Electronic ISSN: 1364-5528
• Publisher: Royal Society of Chemistry
• Peer Reviewed: Peer Reviewed
• Issue: 13
• DOI: https://doi.org/10.1039/c7an02109g
• Public URL: https://keele-repository.worktribe.com/output/1054026
• Publisher URL: https://pubs.rsc.org/en/content/articlelanding/2018/an/c7an02109g