Fluorometric determination of microRNA using arched probe-mediated isothermal exponential amplification combined with DN
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ORIGINAL PAPER
Fluorometric determination of microRNA using arched probe-mediated isothermal exponential amplification combined with DNA-templated silver nanoclusters Hao Wu 1
&
Jun Wu 1 & Yaling Liu 1 & Hongyong Wang 1 & Pei Zou 1
Received: 15 May 2019 / Accepted: 16 September 2019 # Springer-Verlag GmbH Austria, part of Springer Nature 2019
Abstract A highly sensitive fluorometric method is described for the determination of microRNA-141. It is based on the use of arched probe-mediated isothermal exponential amplification reaction (EXPAR) and of DNA-templated silver nanoclusters (DNAAgNCs). The EXPAR utilizes microRNA-141 as the trigger, polymerases and endonucleases as amplification activators, and two arched probes as exponential amplification templates. This enables the conversion of microRNA to a large number of reporter sequences under isothermal conditions within minutes. The generated reporter sequences act as scaffolds for the synthesis of fluorescent DNA-AgNCs by reduction of Ag (I) with NaBH4. The DNA-AgNCs function as signalling fluorophores with excitation/emission maxima at 540/610 nm. The method exhibits high sensitivity for microRNA-141 with a detection limit as low as 0.87 fM and a dynamic range from 1 fM to 500 fM. The method can distinguish nucleotides in the microRNA-200 family. Keywords MicroRNA-141 . Arched probe . Silver nanoclusters . Isothermal amplification . DNA polymerase . Nicking endonuclease . Human serum
Introduction MicroRNAs are short, endogenous, noncoding RNAs with 17~25 nucleotides in length. They are generated by consecutive cleavages of long primary transcripts by two RNase III enzymes (Drosha and Dicer) [1]. They can influence many biological processes including cell proliferation, differentiation and apoptosis by regulating gene expression through translational repression or messenger RNA (mRNA) Hao Wu and Jun Wu contributed equally to this work. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00604-019-3836-4) contains supplementary material, which is available to authorized users. * Hao Wu [email protected] * Pei Zou [email protected] 1
Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi Jiangsu 214063 People’s Republic of China
degradation [2]. Accumulating evidence has revealed that the aberrant expression of microRNAs in human tissues and blood samples is closely related to various diseases, such as diabetes [3], neurological disorders [4] and especially cancers [5]. Hence, accurate and quantitative analysis of microRNA expression is of great significance for the biochemical and clinical studies on pathogenesis and early diagnosis. Considering the importance of microRNA analysis, several conventional techniques have been developed for microRNA expression profiling over the last decades, such as Northern blotting [6], microarrays [7] and reverse transcription polymerase chain reaction (RT-PCR) [8]. Unfortunately, these
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