title: A target-triggered dual amplification strategy for sensitive detection of microRNA.
abstract: The accurate and quantitative analysis of microRNA (miRNA) expression is critical for biomedical research and clinical theranostics. In this study, we report a novel sensor for the sensitive detection of miRNA based on a duplex-specific nuclease (DSN)-assisted dual signal amplification strategy. A chimeric probe (DNA/2-OMe-RNA) that consists of a miRNA recognition DNA sequence and a Taqman probe hybridization RNA sequence (2'-O-methyl RNA) was designed and synthesized. One molecule of target miRNA can trigger cyclical cleavage of the chimeric probes to produce 2'-O-methyl RNA by DSN in the first round of amplification. The 2'-O-methyl RNA molecules can subsequently hybridize with Taqman probes and initiate the second round of cyclical amplification to generate detectable fluorescence by DSN. The proposed strategy exhibits high specificity in discriminating cognate miRNAs, and the dual signal transduction process enables the detection of miRNA concentrations as low as 7.3fM. We further applied this assay to miRNA quantification in cancer cells to confirm its applicability. The present study provides a sensitive, specific and simple method for miRNA detection and holds great potential for further application in biomedical research and in the clinical laboratory.
authors.affiliation: Department of Laboratory Medicine, Nanfang Hospital, Southern Medical University, Guangzhou 510515, Guangdong Province, PR China; Guangdong Engineering and Technology Research Center for Rapid Diagnostic Biosensors, Nanfang Hospital, Southern Medical University, Guangzhou 510515, Guangdong Province, PR China.
authors.forename: Weifeng
authors.initials: W
authors.lastname: Lv
authors.affiliation: Department of Laboratory Medicine, Nanfang Hospital, Southern Medical University, Guangzhou 510515, Guangdong Province, PR China; Guangdong Engineering and Technology Research Center for Rapid Diagnostic Biosensors, Nanfang Hospital, Southern Medical University, Guangzhou 510515, Guangdong Province, PR China.
authors.forename: Jiamin
authors.initials: J
authors.lastname: Zhao
authors.affiliation: Department of Laboratory Medicine, Nanfang Hospital, Southern Medical University, Guangzhou 510515, Guangdong Province, PR China; Guangdong Engineering and Technology Research Center for Rapid Diagnostic Biosensors, Nanfang Hospital, Southern Medical University, Guangzhou 510515, Guangdong Province, PR China.
authors.forename: Bo
authors.initials: B
authors.lastname: Situ
authors.affiliation: Department of Laboratory Medicine, Nanfang Hospital, Southern Medical University, Guangzhou 510515, Guangdong Province, PR China; Guangdong Engineering and Technology Research Center for Rapid Diagnostic Biosensors, Nanfang Hospital, Southern Medical University, Guangzhou 510515, Guangdong Province, PR China.
authors.forename: Bo
authors.initials: B
authors.lastname: Li
authors.affiliation: Department of Laboratory Medicine, Nanfang Hospital, Southern Medical University, Guangzhou 510515, Guangdong Province, PR China; Guangdong Engineering and Technology Research Center for Rapid Diagnostic Biosensors, Nanfang Hospital, Southern Medical University, Guangzhou 510515, Guangdong Province, PR China.
authors.forename: Wen
authors.initials: W
authors.lastname: Ma
authors.affiliation: Department of Laboratory Medicine, Nanfang Hospital, Southern Medical University, Guangzhou 510515, Guangdong Province, PR China; Guangdong Engineering and Technology Research Center for Rapid Diagnostic Biosensors, Nanfang Hospital, Southern Medical University, Guangzhou 510515, Guangdong Province, PR China.
authors.forename: Jumei
authors.initials: J
authors.lastname: Liu
authors.affiliation: Department of Laboratory Medicine, Nanfang Hospital, Southern Medical University, Guangzhou 510515, Guangdong Province, PR China; Guangdong Engineering and Technology Research Center for Rapid Diagnostic Biosensors, Nanfang Hospital, Southern Medical University, Guangzhou 510515, Guangdong Province, PR China.
authors.forename: Zixian
authors.initials: Z
authors.lastname: Wu
authors.affiliation: School of Engineering and Materials Science, Queen Mary University of London, London E1 4NS, UK.
authors.forename: Wen
authors.initials: W
authors.lastname: Wang
authors.affiliation: Research Center of Clinical Medicine, Nanfang Hospital, Southern Medical University, Guangzhou 510515, Guangdong Province, PR China.
authors.forename: Xiaohui
authors.initials: X
authors.lastname: Yan
authors.affiliation: Department of Laboratory Medicine, Nanfang Hospital, Southern Medical University, Guangzhou 510515, Guangdong Province, PR China; Guangdong Engineering and Technology Research Center for Rapid Diagnostic Biosensors, Nanfang Hospital, Southern Medical University, Guangzhou 510515, Guangdong Province, PR China. Electronic address: nfyyzhenglei@smu.edu.cn.
authors.forename: Lei
authors.initials: L
authors.lastname: Zheng
mesh_terms: D015374:Biosensing Techniques / Q000379:methods; D045744:Cell Line, Tumor; D015342:DNA Probes / Q000737:chemistry; D006801:Humans; D059346:Magnets / Q000737:chemistry; D008745:Methylation; D035683:MicroRNAs / Q000032:analysis*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009693:Nucleic Acid Hybridization / Q000379:methods; D015347:RNA Probes / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods
chemical_list: D015342:DNA Probes; D035683:MicroRNAs; D015347:RNA Probes
keywords: Chimeric probes; Dual signal amplification; Duplex-specific nuclease; Magnetic microspheres; MicroRNA