Highly sensitive multiplex detection of microRNA using light-up RNA aptamers

MicroRNAs (miRNA) involved in various biological processes can serve as important biomarkers for early diagnosis and prognosis of various cancers. Quantitative reverse transcription PCR is the gold standard method to detect miRNAs in clinical settings, but it requires expensive equipment and complicated primer designs, thus hindering its use in the field. Herein, we developed a simple, cost-effective strategy for the multiplex detection of miRNA using light-up RNA aptamers as the key detection component. In principle, the presence of target miRNAs produces a large amount of light-up RNA aptamers such as Spinach and Mango aptamers through strand displacement amplification in combination with transcription amplification. Consequently, light-up RNA aptamers bind to their corresponding fluorogens and emit highly enhanced fluorescence signals at different wavelengths, which enables the multiplex detection of target miRNAs. With the proposed strategy, target miRNAs such as miR21 and miR141 were simultaneously determined with limits of detection of 0.955 fM and 0.195 fM, respectively. This assay was successfully applied for the quantification of miRNAs in different cell lines, demonstrating its practical utility in clinical diagnosis and prognosis.