A highly sensitive and selective signal-on strategy for microRNA quantification

• Published in: Analytica chimica acta Vol. 1100; pp. 258 - 266
• Main Authors: Pan, Li, Zhang, Huaisheng, Zhao, Jingjin, Li, Xiangtang, Xu, Rui, Mo, Yinyuan, Tchounwou, Paul B., Liu, Yi-Ming
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.03.2020
• Subjects: Amplified fluorescence signal; Animals; Biological Assay; Biological samples; Calibration; Cattle; Cell Culture Techniques; Chromatography, High Pressure Liquid - methods; DNA, Single-Stranded - genetics; HPLC quantification; Humans; Limit of Detection; Low background detection; MCF-7 Cells; microRNAs; MicroRNAs - analysis; MicroRNAs - genetics; Nucleic Acid Amplification Techniques; Polyadenylation; microRNAs; Biological samples; Amplified fluorescence signal; HPLC quantification; Polyadenylation; Low background detection
• ISSN: 0003-2670; 1873-4324
• DOI: 10.1016/j.aca.2019.12.003

MicroRNAs (miRNAs) are associated with physiological and pathological processes. They are recognized as biomarkers for diseases diagnosis and treatment evaluation. Herein we propose a simple and cost-effective HPLC method for quantitative assay of target miRNAs with femtomolar sensitivity, single-base discrimination selectivity and low background. The assay is based on an innovative signal-on strategy. In this strategy, polyadenylation of poly(A) polymerase extends an all ‘A’ sequence at the end of target miRNA, and the substantially increased number of adenine bases are labeled with 2-Chloroacetaldehyde (CAA) to open a signal-on mode and realize a signal amplification. The linearly amplified fluorescence signal is separated from other inference signals and quantified by high performance liquid chromatography with fluorescence detection (HPLC-FD). Combining with affinity magnetic solid phase extraction (MSPE), the method is well suited for analysis of complex biological samples such as serum and cell lysate with nearly zero background fluorescence. Taking miRNA-21 as the model analyte, this absolute quantification method has a limit of detection of 200 fM and a linear calibration curve (R2 = 0.999) in the range from 2.00 pM to 1.00 nM. Using locked nucleic acid (LNA) modified probes rather than ssDNA probes, the assay selectivity is improved. Moreover, analysis of bovine serum and cell lysate samples by using the method is demonstrated. Intracellular content of miRNA-21 is found to be 0.0150 amol/cell in MCF-7 cells with an assay repeatability of 4.0% (RSD, n = 3). The present HPLC quantification of miRNA offers an accurate, reliable, and cost-effective means for quantitative assay of miRNAs occurring in biological samples. Also importantly, it eliminates the need for total RNA isolation for the analysis. It may be useful for more effective diagnosis of diseases and therapeutic evaluation. Quantitative assay of target miRNAs by HPLC with femtomolar sensitivity. [Display omitted] •An HPLC method for miRNA quantification with femtomolar sensitivity and single-base discrimination selectivity is reported.•Advantages of the proposed method include a low fluorescence background and linear signal-concentration calibration curves.•This is the first HPLC assay for quantifying microRNAs based on isothermal signal amplification.•Real-life samples including serum and cell culture were analyzed without any sample pretreatments.