Direct recognition and sensitive detection of circular RNA with ligation-based PCR

Abstract

Circular RNAs (circRNAs), which function as a kind of newly discovered biomarker, have been shown to play an important role in gene expression, signaling pathway and disease diagnosis. Accurate recognition and sensitive detection of sequence-specific circRNA of interest can advance our understanding of its biological functions and the diagnosis of human diseases. Herein, we have newly developed a highly sensitive and specific method to quantitatively detect circRNA through accurate ligation of two ingeniously designed DNA probes using splintR ligase at the junction site of circRNA, which can directly discriminate circRNA from the corresponding linear RNA. With PCR amplification of the ligated DNA probes, circRNA as low as 1 fM can be detected with the dynamic range spanning over five orders of magnitude. This method has been successfully applied to the detection of circRNA in total RNA samples extracted from cell lines.