PCR-Free Detection of Long Non-Coding HOTAIR RNA in Ovarian Cancer Cell Lines and Plasma Samples

Abstract

Long non-coding RNA HOX transcript antisense intergenic RNA (HOTAIR) is one of the promising biomarkers that has widely been used in determining the stages of many cancers, including ovarian cancer. In cancer diagnostics, the two key analytical challenges for detecting long non-coding RNA biomarkers are i) the low concentration levels (nM to fM range) in which they are found and ii) the analytical method where broad dynamic range is required (four to six orders of magnitude) due to the large variation in expression levels for different HOTAIR RNAs. To meet these challenges, we report on a biosensing platform for the visual (colorimetric) estimation and subsequent electrochemical quantification of ovarian-cancer-specific HOTAIR using a screen-printed gold electrode (SPE-Au). Our assay utilizes a two-step strategy that involves (i) magnetic isolation and purification of target HOTAIR sequences and (ii) subsequent detection of isolated sequences using a sandwich hybridization coupled with horseradish peroxidase (HRP)-catalyzed reaction of 3,3′,5,5′-tetramethylbenzidine (TMB) in the presence of hydrogen peroxide. The assay achieved a detection limit of 1.0 fM HOTAIR in spiked buffer samples with excellent reproducibility (% RSD ≤ 5%, for n = 3). It was successfully applied to detect HOTAIR in cancer cell lines and a panel of plasma samples derived from patients with ovarian cancer. The analytical performance of the method was validated with standard RT-qPCR. We believe that the proof of concept assay reported here may find potential use in routine clinical settings for the screening of cancer-related lncRNAs.

Electrochemical Detection of Global DNA Methylation Using Biologically Assembled Polymer Beads

“…Advanced stage at diagnosis, poor prognosis and high incidence of resistance to therapy account for the most critical hurdles for ovarian cancer patients. Recently, our group has developed an amplification-free platform for the naked-eye observation and electrochemical quantification of long non-coding RNAs in ovarian cancer patients [16]. DNA methylation is another biomarker that plays a vital role in ovarian cancer, with several tumour suppressor genes shown to be hypermethylated [17].…”

(In)Distinctive Role of Long Non-Coding RNAs in Common and Rare Ovarian Cancers

“…A peptide nucleic acid (PNA) designed to specifically block the activity of HOTAIR has been successful in reducing ovarian tumor growth in vitro and in vivo, and improved survival of xenograft mice, and has been suggested as a potential new therapeutic approach for the treatment of OC [170]. The potential use of this lncRNA as a biomarker has led to the development of various methods for detection of this lncRNA from samples derived from patients with OC, primarily plasma [171,172].…”

“…This is especially true for lncRNAs encoded by a single exon, such as MALAT1 [251]. Soda et al, have recently demonstrated that HOTAIR can be detected from plasma samples using electrochemical detection, and they propose that this assay could be used in a clinical setting for the detection of various lncRNA biomarkers [172]. It is also possible to detect microproteins encoded by lncRNAs in the extracellular vesicles in human plasma samples, and these microproteins could be used as diagnostic markers [252].…”

Microneedle Arrays for Sampling and Sensing Skin Interstitial Fluid

“…Alternatively, microfluidic technology can address the limitations associated with conventional ISF sampling techniques. Microfluidics is an emerging science and technology that can offer significant improvements over various fields, including surface science [15], porous systems [16], nanotechnology for disease diagnosis [17], mixing [18], and separation [19,20], mechanobiology [21], cancer research [22,23], cell culture [24,25], single-cell analysis [26], drug delivery at cellular [27] and tissue levels [28], electrochemical biosensing [29], and POC sensing [30]. Furthermore, the emerging field of micro elastofluidics can provide microfluidic solutions for a flexible, conformal system attached to the skin [27].…”