RNA amplification has extensive applications on biochemistry and its related fields. Here, we present an isothermal strategy named rolling circle reverse transcription-mediated RNA amplification (RCRT-MRA) to amplify small RNAs with accurate length and sequence. The target RNA and complementary DNA were circularized to serve as amplicons replicated via the rolling circle mechanism. The transcription product consisting of tandemly repeated RNA units, was monomerized by site-specific cleavage to generate amplified RNA with authentic length and sequence. T4 DNA ligase was chosen to circularize RNA template for its high efficiency and low cost. SuperScript IV reverse transcriptase was found to be able to catalyze the RCRT reaction on the circular RNA template, and the reaction efficiency was enhanced by adding the nicking enzyme, Nb.BbvCI to the RCRT system. E. Coli RNA polymerase, instead of the commonly used T7 RNA polymerase, was applied to synthesize long-strand RNA product for its high universality and processivity. Under the optimized conditions, small RNAs can be precisely amplified by 10^5∼6 folds. The fidelity of the established method was demonstrated by the accordance of the sequencing result and the initial RNA sequences. Free from expensive thermal cycler (necessary for RT-PCR-based amplification), precise replication of the initial RNA and high fidelity will enable the established strategy to be applied in RNA-seq, mRNA profiling, microarray analysis and RNA-based SELEX as well.

RNA扩增在生物化学及其相关领域具有广泛的应用。在这里，我们提出了一种等温策略，称为滚动环逆转录介导的RNA扩增（RCRT-MRA），以扩增具有正确长度和序列的小RNA。将目标RNA和互补DNA环化以用作通过滚环机制复制的扩增子。通过位点特异性切割使由串联重复的RNA单元组成的转录产物单体化，以生成具有真实长度和序列的扩增RNA。选择T4 DNA连接酶以使其高效且低成本来环化RNA模板。发现SuperScript IV逆转录酶能够催化环状RNA模板上的RCRT反应，并且通过添加切刻酶Nb可以提高反应效率。E. Coli RNA聚合酶代替了常用的T7 RNA聚合酶，由于其高通用性和可加工性而被用于合成长链RNA产物。在优化的条件下，小RNA可以精确扩增10 ^5〜6倍。根据测序结果和初始RNA序列证明了所建立方法的保真度。无需昂贵的热循环仪（基于RT-PCR的扩增所必需），初始RNA的精确复制和高保真度将使既定策略可应用于RNA-seq，mRNA分析，微阵列分析和基于RNA的SELEX 。