Sensitive detection of mRNA by using specific cleavage-mediated isothermal exponential amplification reaction

Abstract

Messenger RNA (mRNA) as a bridge links the organism’s genotypes to its phenotypes. Quantitative detection of mRNA is vital in basic studies of life science and medical diagnostics. In this work, a sensitive, specific, and reverse transcription-free approach for quantification of mRNA based on cleavage-mediated isothermal exponential amplification reaction (IEXPAR) is established. The target mRNA firstly hybridizes with the flexibly designed DNA probe to form a loop structure. RNase H specifically digests the hybridized mRNA and releases the loop part of the mRNA, which can serve as the trigger to initiate IEXPAR. In this design, the DNA probe and the IEXPAR templates can specifically recognize three regions on the target mRNA, so that the high specificity can be achieved. Moreover, the specificity of RNase H can avoid the contamination of genomic DNA. IEXPAR is a powerful nucleic acid amplification technique with rapid, simple and cost-effective features, making the proposed assay highly sensitive and fast. With this assay, as low as 100 zmol target mRNA can be quantitatively detected and wide linear range with six orders of magnitude can be obtained. The assay can also be successfully applied to determine p53 mRNA in 5ng total RNA sample extracted from MCF-7 breast cancer cells.