文献类型：期刊文献

英文题名：Sensitive Detection of MicroRNA in Complex Biological Samples via Enzymatic Signal Amplification Using DNA Polymerase Coupled with Nicking Endonuclease

作者：Yin, Bin-Cheng[1];Liu, Yu-Qiang[1];Ye, Bang-Ce[1]

机构：[1]E China Univ Sci & Technol, Lab Biosyst & Microanal, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China

年份：2013

卷号：85

期号：23

起止页码：11487

外文期刊名：ANALYTICAL CHEMISTRY

收录：;EI(收录号：20135017072101);WOS:【SCI-EXPANDED(收录号:WOS:000327999800045)】；

基金：This work was jointly supported by the National Natural Science Foundation of China (Grant Nos. 21335003, 21205040, and 21075040), the Shanghai Fund (12ZR1442700, 11 nm0502500), the Fundamental Research Funds for the Central Universities, and Hitachi, Ltd.

语种：英文

外文关键词：RNA - Clinical research - Polymerization - Polymerase chain reaction - Diagnosis - Diseases - Isotherms - Signal transduction

摘要：MicroRNA (miRNA) has become an ideal biomarker candidate for cancer diagnosis, prognosis, and therapy. In this study, we have developed a novel one-step method for sensitive and specific miRNA detection via enzymatic signal amplification and demonstrated its practical application in biological samples. The proposed signal amplification strategy is an integrated "biological circuit" designed to initiate a cascade of enzymatic polymerization reactions in order to detect, amplify, and measure a specific miRNA sequence by using the isothermal strand-displacement property of a mesophilic DNA polymerase together with the nicking activity of a restriction endonuclease. The circuit is composed of two molecular switches operating in series: the nicking endonuclease-assisted isothermal polymerization reaction activated by a specific miRNA and the strand-displacement polymerization reaction designed to initiate molecular beacon-assisted amplification and signal transduction. The hsa-miR-141 (miR-141) was chosen as a target miRNA because its level specifically elevates in prostate cancer. The proposed method allowed quantitative sequence-specific detection of miR-141 in a dynamic range from 1 fM to 100 nM, with an excellent ability to discriminate differences among miR-200 family members. Moreover, the detection assay was applied to quantify miR-141 in cancerous cell lysates. The results are in excellent agreement with those from the reverse transcription polymerase chain reaction method. 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.