Ultrasensitive detection of circular RNA by accurate recognition of the specific junction site using stem-loop primer...

Circular RNAs (circRNAs), as a class of newly emerging biomarkers, have shown to be associated with many fundamental life processes and diseases, especially cancer. However, various limitations in currently available detection methods have seriously restricted the development of the studies associated with circRNA's biological functions and the diagnosis of diseases by using circRNA as the biomarker. By specifically designing a pair of stem-loop primers (SLPs) exactly recognize the junction sequence of circRNA, we firstly establish a SLP induced double exponential amplification method for sensitive and specific detection of circRNA with the ability to directly discriminate circRNA from linear RNA. Through the extension of SLPs during thermo cycles, one circRNA can generate a large amount of double stem-loop structure DNA which can initiate the subsequent isothermal amplification, leading to the sensitive detection of as low as 10 aM circRNA which is the most sensitive method for circRNA detection up to now. The proposed method has successfully applied to the detection of circRNA in many kinds of cancer cells in homogeneous solution without any separation step.