Discriminative identification of miRNA let-7 family members with high specificity and sensitivity using rolling circle amplification.

Abstract

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.