利用滚环扩增鉴别同源家族miRNAs

滚环扩增（rolling circle amplification, RCA）是一种基于病毒DNA复制而发明的新技术。近些年，RCA技术已经被广泛应用于微小核糖核酸（micro ribonucleic acid, miRNA）的检测。在miRNA检测研究领域中，鉴别高度同源的家族miRNAs成为该研究领域的瓶颈。本研究引入新型的RCA技术来增加鉴别的灵敏度和特异性，进一步提高家族miRNA鉴别的灵敏度，滚环扩增的程度用相对荧光强度来表示。研究结果显示，T4 RNA连接酶2可在RCA的环化过程中实现最大的环化效率，从而提高RCA的检测特异性。本文利用优化的RCA技术，实现对let 7高度同源的家族miRNAs高灵敏度的鉴别，灵敏度可达5 fmol。let 7a的滚环探针对Let 7a这一miRNA扩增后的相对荧光强度为1 550，而对其他的家族miRNA相对荧光强度仅为260。其他的家族miRNA探针在鉴别时相对荧光强度也显示了较大的差异。而依靠传统的RT-qPCR方法的鉴别灵敏度是4 pmol，与本研究相比，灵敏度低了近1 000倍。本研究的结果表明，利用RCA技术鉴别高度同源性miRNAs是高效灵敏的，此前未见相关研究的报道。RCA技术可能被应用于miRNA高灵敏度检测和鉴别的相关研究中。

A New Technique to Discriminate Homologous microRNAs Based on Rolling Circle Amplification

Rolling circle amplification (RCA) is a newly invented technology based on the virus DNA reproduction. Recent years, it was widely used in the field of miRNA detection. Discrimination of highly homologous miRNAs is a choke point during the research of miRNA. We creatively imported the molecular beacon as a model to optimize the RCA process to increase the specificity and sensitivity of miRNA discrimination. Relative fluorescence intensity (RFI) was monitored as the fluorescence changes in RCA process. Results showed that T4 RNA ligase 2 could access the highest efficiency of circularization during the RCA process which promoted RCA at high specificity. By using RCA technology, members of highly homologous miRNAs Let 7 could be discriminated at the amount of 5 fmol. The RFI of Let 7a is about 1 550, whereas RFI of other family member is about 260. Discriminating data of other Let 7 miRNA members also showed significant differences. This sensitivity of discriminating miRNAs could not be achieved by traditional RT-qPCR method, which discreminating sensitivity is 4 pmol. Optimal RCA technology used in this study in discriminating highly homologous family miRNAs has not been reported before. Results and conclusions obtained in the study can promote the applications of RCA in the field of high-sensitivity detection of miRNAs.