A novel isothermal method using rolling circle reverse transcription for accurate amplification of small RNA sequences.

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 105∼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.