Ultrasensitive Detection of microRNAs by Exponential Isothermal Amplification
Ultrasensitive Detection of microRNAs by Exponential Isothermal Amplification
复制标题
通过指数等温扩增超灵敏检测 microRNA
DOI：
10.1002/anie.201001375
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发表时间：
2010-01-01
影响因子：
16.6
通讯作者：
Cheng, Yongqiang
中科院分区：
文献类型：
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作者：
Jia, Hongxia;Li, Zhengping;Cheng, Yongqiang
microRNA（miRNAs）是一类内源性的非编码小RNA分子（19-23个核苷酸（nt））。通过与靶信使RNA形成RNA诱导的沉默复合物（RISC）的翻译抑制或靶降解，miRNA在广泛的生物过程中发挥重要作用，包括增殖、发育、代谢、免疫应答、肿瘤发生和病毒感染。[1]近年来，miRNAs的生物学功能已成为研究热点。miRNA的检测对于更好地理解这些生物分子的功能至关重要，并且对于人类疾病的早期诊断以及通过使用miRNA作为靶标发现新药具有巨大潜力。[2]Northern-blotting分析现在被认为是检测miRNA的标准方法。[3]微阵列由于其高通量筛选能力而越来越多地用于miRNA表达分析。[4]然而，这些方法的灵敏度和特异性并不令人满意，因为小尺寸，序列相似性和低丰度的miRNA。[5，6]已经报道了用于miRNA分析的各种扩增策略，以提高该方法的灵敏度和特异性，例如实时PCR [7]，改良的入侵者测定法[8]，核酶扩增[9]，纳米颗粒扩增方法[10]，滚环扩增[11]和基于缀合聚合物的方法。[12]在这些方法中，实时PCR是最敏感和实用的。然而，短长度的miRNAs使得PCR设计非常复杂。必须使用茎环DNA探针、LNA修饰的DNA探针（LNA=锁核酸）或双荧光标记的TaqMan探针，这些探针大大增加了实验成本和复杂性。因此，需要一种简单、低成本和高灵敏度的miRNA检测方法。
MicroRNAs (miRNAs) are a class of endogenous, noncoding small RNA molecules (19–23 nucleotides (nt)). Through translational repression or target degradation by the formation of an RNA-induced silencing complex (RISC) with target messenger RNAs, miRNAs play important roles in a wide range of biological processes, including proliferation, development, metabolism, immunological response, tumorigenesis, and viral infection.[1] Recently, the biological functions of miRNAs have become an area of intense investigation. The detection of miRNAs is imperative for gaining a better understanding of the functions of these biomolecules and has great potential for the early diagnosis of human disease as well as the discovery of new drugs through the use of miRNAs as targets.[2]Northern-blotting analysis is now considered the standard method for miRNA detection.[3] Microarrays are being used more and more for miRNA-expression analysis as a result of their high-throughput-screening capability.[4] However, the sensitivity and specificity of these methods are not satisfactory because of the small size, sequence similarity, and low abundance of miRNAs.[5, 6] Various amplification strategies for miRNA analysis have been reported to improve the sensitivity and specificity of the approach, such as real-time PCR,[7] the modified invader assay,[8] ribozyme amplification,[9] nanoparticle amplification methods,[10] rolling circle amplification,[11] and conjugated-polymer-based methods.[12] Among these methods, real-time PCR is the most sensitive and practical. However, the short length of miRNAs makes the PCR design very sophisticated. Stem–loop DNA probes, LNA-modified DNA probes (LNA= locked nucleic acid), or doubly fluorescence labeled TaqMan probes have to be used, and these probes greatly increase the experimental cost and complexity. Therefore, a simple, low-cost, and highly sensitive method for miRNA detection is desirable.