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

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.