title: Sensitive detection of microRNA in complex biological samples by using two stages DSN-assisted target recycling signal amplification method.
abstract: MicroRNA (miRNA) has become an important biomarker candidate for cancer diagnosis, prognosis, and therapy. In this study, we have developed a novel fluorescence method for sensitive and specific miRNA detection via duplex specific nuclease (DSN) signal amplification and demonstrated its practical application in biological samples. Malachite green (MG) was employed as a "label-free" signal transducer since fluorescence of MG could be enhanced by 100-fold when MG were binding to a G-quadruplex structure formed within the d(G2T)13G sequence. The proposed signal amplification strategy is an integrated "biological circuit" designed to initiate a cascade of enzymatic reactions in order to detect, amplify, and measure a specific miRNA sequence by using the isothermal cleavage property of a DSN. The circuit is composed of two molecular switches operating in series: the amplification reaction activated by a specific miRNA and the strand-displacement polymerization reaction designed to initiate molecular beacon-assisted amplification and signal transduction by using MG/G-quadruplex complex. The hsa-miR-141 (miR141) was chosen as a target miRNA because its level specifically abnormal in a wide range of common human cancers including breast, lung, colon, and prostate cancer. The proposed method allowed quantitative sequence-specific detection of miR141 (with a detection limit of 1.03pM) in a dynamic range from 1pM to 10μM, with an excellent ability to discriminate differences in miRNAs. Moreover, the detection assay was applied to quantify miR141 in cancerous cell lysates. On the basis of these findings, we believe that this proposed sensitive and specific assay has great potential as a miRNA quantification method for use in biomedical research and clinical diagnosis.
authors: Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi, Jiangsu 214063, China. Electronic address: zhangkai@jsinm.org.
authors: Kai
authors: K
authors: Zhang
authors: Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi, Jiangsu 214063, China.
authors: Ke
authors: K
authors: Wang
authors: Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi, Jiangsu 214063, China.
authors: Xue
authors: X
authors: Zhu
authors: Department of Laboratory Medicine, Wuxi Municipal Women and Children Health Hospital, Wuxi, Jiangsu 214000, China.
authors: Fei
authors: F
authors: Xu
authors: Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi, Jiangsu 214063, China.
authors: Minhao
authors: M
authors: Xie
mesh_terms: D000818:Animals; D033381:Anomura / Q000201:enzymology; D015374:Biosensing Techniques / Q000379:methods; D004396:Coloring Agents / Q000737:chemistry; D015342:DNA Probes / Q000737:chemistry / Q000378:metabolism; D004720:Endonucleases / Q000378:metabolism; D054856:G-Quadruplexes; D056945:Hep G2 Cells; D006801:Humans; D057230:Limit of Detection; D035683:MicroRNAs / Q000032:analysis* / Q000378:metabolism; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012394:Rosaniline Dyes / Q000737:chemistry
chemical_list: D004396:Coloring Agents; D015342:DNA Probes; C533895:MIRN141 microRNA, human; D035683:MicroRNAs; D012394:Rosaniline Dyes; C005095:malachite green; D004720:Endonucleases
keywords: Amplification method; Cell lysates; DSN; Malachite green; MiRNA