无标记microRNA芯片分析方法的建立与优化  

Establishment and Optimization of a Label-free microRNA Microarray Analytical Method

作　　者：段德民[1] 惠利省[2] 李丽诗[2] 阎锡蕴[1] 李炯[2] 

机构地区：[1]中国科学院生物物理研究所,北京100101 [2]中国科学院苏州纳米技术与纳米仿生研究所,苏州215123

出　　处：《生物化学与生物物理进展》2013年第5期479-489,共11页Progress In Biochemistry and Biophysics

基　　金：国家自然科学基金(31270908);中国博士后科学基金(2012M510049;20110491461);中国科学院王宽诚博士后工作奖励基金;江苏省自然科学基金(BK2011361);江苏省博士后科学基金(1101052C)资助项目~~

摘　　要：MicroRNAs(miRNAs)是生物体内源的一类非编码小分子RNA,它与癌症的发生息息相关,是一个有潜质的生物标志物,细胞的发育、分化、增殖、凋亡都与miRNAs调控有关.miRNAs研究中关键环节是其表达谱的分析,用芯片分析miRNAs表达谱,通常要在杂交前对样品进行分离、标记、纯化,这是整个检测过程中最耗时耗力、费用昂贵的一个步骤,且在此过程中由于酶的使用及步骤的增加还可能改变样品中目标序列的初始比例,影响实验结果的可靠性.为解决这些问题,作者建立了一种新型的"无标记microRNA芯片分析"方法,该技术基于堆积杂交(stacking hybridization)原理,引入一段预先标记荧光的通用标签序列(universal tag,UT),因而被命名为SHUT检测方法.本文主要对SHUT assay的整个实验过程进行了系统优化,并对其灵敏度和特异性等相关指标进行了评价.实验结果表明:该新型芯片技术检测灵敏度可达2 fmol/L,不但可以区分只有1个碱基差异的miRNAs家族成员,还可以有效排除非活性pri-miRNA与pre-miRNA前体的交叉杂交信号,而且100 ng的总RNA即可用于检测分析;该新型芯片技术对于miRNAs及其他短链核酸分子的检测分析是一个理想快速的检测平台.MicroRNAs （miRNAs） are a class of short, endogenous, non-coding small RNAs. The regulation function accompanies with the cell growth, differentiation, proliferation and apoptosis. The miRNAs expression is closely related with the development of cancer and has been considered as a potential biomarker. The analysis of the miRNAs expression profile is a critical part in the studying ofmiRNAs. Usually, miRNAs in biological samples require fraction, labeling and purification before applied to most assays. This is the most time-consuming, labor-intensive and the highest cost in the assay process. The assay results would be affected for the initial ratio of the sample target miRNAs which may have been changed due to the application of enzymes and complicated steps in this preprocess. The main purpose of this work is to develop a new microRNA microarray platform that free of the sample labeling. It is named the stacking hybridization-based universal tag （SHUT） assay for it takes advantage of stacking hybridization interaction and involves a universal tag （UT） an 8mers oligonucleotide labeled previously. This article focuses on the optimization of the experimental procedures in the SHUT assay and the evaluation of its properties, such as the sensitivity and the specificity. The results show that the microarray has the advantage of high sensitivity, that even 2 fmol/L input miRNAs can be detected. The miRNAs microarray has perfect selectivity simultaneously. It can distinguish the target miRNAs from other family members with only one base mismatched. In particularly, the pri-miRNA and pre-miRNA can be easily discriminated from mature-miRNAs, which enables as little as 100 ng total RNAs to be analyzed directly and rapidly. All these results have shown that the application of this new microarray platform approach is a rapid and ideal platform for the detection as well as analysis ofmiRNAs and other small nucleic acid molecules.

关 键 词：堆积杂交 通用标签 microRNAs芯片 表达谱 

分 类 号：Q5[生物学—生物化学] Q7