Sensitive Quantification of MicroRNAs by Isothermal Helicase-Dependent Amplification,Analytical Chemistry

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Sensitive Quantification of MicroRNAs by Isothermal Helicase-Dependent Amplification
Analytical Chemistry ( IF 6.7 ) Pub Date : 2017-05-11 00:00:00 , DOI: 10.1021/acs.analchem.7b01113
Fei Ma ₁ , Meng Liu ₁ , Bo Tang ₁ , Chun-yang Zhang ₁

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Dysregulation of microRNA expression levels is closely associated with a variety of human diseases, and their rapid and sensitive quantification is essential to clinical diagnosis and therapy. Because of their poor sensitivity, conventional quantification methods are unable to detect low-abundance microRNAs. Alternatively, nucleic acid amplification approaches have been introduced to improve the detection sensitivity, but most of them involve complicated probe design and time-consuming procedures. Herein, we report a simple, rapid, and sensitive fluorescent method for label-free detection of low-abundance microRNAs based on isothermal helicase-dependent amplification. In this assay, the target microRNA may specifically hybridize with the 3′-terminus of the linear probe to form a DNA-microRNA heteroduplex, protecting the probes from exonuclease I digestion. The remaining probes may be subsequently amplified by helicase-dependent amplification, generating an ultrahigh fluorescence signal within 30 min. This assay is very sensitive with a low detection limit of 12.8 fM and exhibits a large dynamical range from 100 fM to 10 nM. Moreover, this assay can discriminate different microRNA family members, and it can be used to absolutely quantify endogenous microRNA of total RNA samples extracted from cancer cells, providing a powerful tool for biomedical research and clinical diagnostics.

中文翻译：

通过等温解旋酶依赖性扩增对MicroRNA进行灵敏定量

microRNA表达水平的失调与多种人类疾病密切相关，其快速灵敏的定量对于临床诊断和治疗至关重要。由于灵敏度低，常规定量方法无法检测低丰度的microRNA。或者，已引入核酸扩增方法来提高检测灵敏度，但是大多数方法都涉及复杂的探针设计和耗时的过程。在本文中，我们报告了一种基于等温解旋酶依赖性扩增的简单，快速，灵敏的荧光方法，用于无标签的低丰度microRNA的检测。在此分析中，目标microRNA可以与线性探针的3'末端特异性杂交，以形成DNA-microRNA异源双链体，保护探针免受核酸外切酶I的消化。随后可以通过解旋酶依赖性扩增来扩增其余的探针，从而在30分钟内产生超高荧光信号。该测定法非常灵敏，检测限低至12.8 fM，并具有100 fM至10 nM的大动态范围。而且，该测定法可以区分不同的microRNA家族成员，并且可以用于绝对定量从癌细胞中提取的总RNA样品的内源性microRNA，为生物医学研究和临床诊断提供了有力的工具。8 fM的动态范围从100 fM到10 nM。而且，该测定法可以区分不同的microRNA家族成员，并且可以用于绝对定量从癌细胞中提取的总RNA样品的内源性microRNA，为生物医学研究和临床诊断提供了有力的工具。8 fM的动态范围从100 fM到10 nM。而且，该测定法可以区分不同的microRNA家族成员，并且可以用于绝对定量从癌细胞中提取的总RNA样品的内源性microRNA，为生物医学研究和临床诊断提供了有力的工具。

更新日期：2017-05-19

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