Multiple stem-loop primers induced cascaded loop-mediated isothermal amplification for direct recognition and specific detection of circular RNAs

Abstract

Circular RNAs (circRNAs), as a novel class of endogenous noncoding RNAs, play important roles in many biological functions, such as acting as microRNA sponges and cancer biomarkers. Rapid and sensitive detection of circRNAs will be of great benefit to better understand their biological functions especially in clinical diagnosis using circRNAs as biomarkers. Herein, by using multiple stem-loop primers (SLPs) to specifically recognize the unique junction site of circRNAs, we demonstrate a multiple SLPs-induced cascaded loop-mediated isothermal amplification (LAMP) assay which has the ability to directly and specifically discriminate circRNAs from homologous linear RNAs without any linear RNA removal procedure. Taking advantage of the displacement activity of Bst DNA polymerase, the rationally designed SLPs can directly recognize the target circRNA and generate a large amount of double stem-loop DNAs which can initiate a cascaded LAMP reaction with improved efficiency. The use of additional SLPs can accelerate the reaction rate; as a result, the LAMP reaction can be finished within 35 min which is much quicker than traditional LAMP. The proposed method can detect as low as 1 fM circRNA and has been successfully applied to the detection of circRNAs in total RNA samples extracted from cell lines.