Highly specific quantification of mRNA mutation in single cells based on RNase H cleavage-assisted reverse transcription (RT)-PCR

Accurate quantitation of site-specific mRNA mutation in single cells or in peripheral blood is of great significance for both biological and biomedical studies. How to eliminate the false-positive interference from the abundant normal mRNA is still a big challenge. Herein, we have proposed an LNA (locked nucleic acid)-assisted high-specificity strategy which can selectively guide the RNase H to cleave only the wild-type mRNA (wtRNA) while the mutant mRNA (mutRNA) will remain intact. The intact mutRNA can be amplified and detected by real-time reverse transcription (RT)-PCR but the disconnected wtRNA will be not replicated at all. Based on the highly selective depletion of wtRNA, this elegant design effectively avoids the false-positive interference from the high background of normal mRNA and thus can guarantee the accurate and reliable detection of rare mutRNA in real biomedical samples. Besides for the excellent specificity, ultrahigh sensitivity is also achieved for this proposed assay, which allows the quantification of mutRNA at single molecule and single cell level. Due to its easy design, high sensitivity and specificity, the established LNA probe-assisted RT-PCR strategy provides a powerful tool for studying the function of mutRNA at the single cell level and for the mutRNA-associated liquid biopsy. (C) 2019 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences. Published by Elsevier B.V. All rights reserved.