Real-time and rapid quantification of microRNAs in cells and tissues using target-recycled enzyme-free amplification strategy

Abstract In this study, a real-time target-recycled enzyme-free amplification strategy-based test (Trefas test) was developed for rapid, simple, isothermal, and highly sensitive microRNA (miRNA) detection. The Trefas relies on rationally designed sequence-specific hairpins (HPs, HP1 and HP2) and the strand displacement process completely free of environment-susceptible enzymes, enhancing the stability and reproducibility of the test. In the absence of target miRNA, the HP2, modified with a fluorophore and a quencher, maintains stem-loop structure so that the fluorescent signal is quenched. However, in the presence of target miRNA, the target miRNA is repeatedly used to trigger continuous HP1-HP2 hybridizations, restoring fluorescence due to the opening of HP2. The developed miR-21 real-time Trefas test exhibited a broad linear dynamic range of 1 pM to 1 μM and a detection limit of 0.58 pM for miR-21 detection in vitro. In particular, the high specificity of the developed miR-21 real-time Trefas test was prominently exhibited by discriminating single base differences in miRNA sequences. Finally, the expression level of miR-21 in the cell lines and clinical tissues was evaluated by the developed miR-21 real-time Trefas test, and the detection results were highly consistent with the results obtained by stem-loop RT-PCR. In summary, our developed test exhibited great potential for further application in biomedical research and early clinical diagnosis.