Zhao Bin, Song Jirui, Guan Yifu
Key Laboratory of National Sport Bureau, Department of Human Movement Sciences, Shenyang Sport University, Shenyang 110102, China.
Key Laboratory of Medical Cell Biology (Ministry of Education), Department of Biochemistry and Molecular Biology, China Medical University, Shenyang 110001, China
Acta Biochim Biophys Sin (Shanghai). 2015 Feb;47(2):130-6. doi: 10.1093/abbs/gmu121. Epub 2014 Dec 22.
Rolling circle amplification (RCA) is a new method based on virus DNA reproduction, which has been widely used in the field of miRNA detection. However, discrimination of highly homologous miRNAs is a bottleneck in the research of miRNA. In this study, the RCA process was creatively used to conduct the discrimination of miRNAs. Results showed that T4 RNA ligase 2 could reach the highest circularization efficiency during the RCA process with higher specificity. By using RCA technology, a member of highly homologous miRNAs, let-7, could be discriminated at the amount of 2.5 fmol. This sensitivity could not be achieved by using traditional reverse transcription quantitative polymerase chain reaction (RT-qPCR) method. In addition, detection of miRNAs by using RCA could reach the amount limit of fmol with a good linearity. Optimal RCA technology used in this study is better than RT-qPCR in discriminating highly homologous family miRNAs. Results from this study can promote the applications of RCA in clinical diagnosis, environment protection, health care, disease inspection and prevention, and national security.
滚环扩增（RCA）是一种基于病毒DNA复制的新方法，已在miRNA检测领域得到广泛应用。然而，高同源性miRNA的鉴别是miRNA研究中的一个瓶颈。在本研究中，创造性地利用RCA过程对miRNA进行鉴别。结果表明，T4 RNA连接酶2在RCA过程中可达到最高的环化效率，且特异性更高。通过使用RCA技术，在2.5 fmol的量下能够鉴别出高同源性miRNA家族中的一个成员let-7。而使用传统的逆转录定量聚合酶链反应（RT-qPCR）方法则无法达到这种灵敏度。此外，利用RCA检测miRNA可达到fmol级的量限，且具有良好的线性。本研究中使用的优化RCA技术在鉴别高同源性家族miRNA方面优于RT-qPCR。本研究结果可推动RCA在临床诊断、环境保护、医疗保健、疾病检测与预防以及国家安全等方面的应用。
