Absolute Quantification of CircRNA Using Digital Reverse Transcription-Hyperbranched Rolling Circle Amplification

Circular RNA (circRNA) is a novel class of non-coding RNA that is a promising biomarker for the diagnosis and prognosis of diseases, such as cancer. Sensitive and quantitative detection of circRNA is of particular significance. However, conventional qRT-PCR is not appropriate for accurate quantification due to the strand-substitution potential during the reverse transcription, which has severely hindered the studies of circRNA. Here, we present a sensitive assay for the detection of circRNA using reverse transcription-hyperbranched rolling circle amplification (RT-HRCA). We further developed droplet digital RT-HRCA (ddRT-HRCA) for circRNA quantification using the microfluidic device. We demonstrate the ddRT-HRCA, modeled on circHIPK3, can achieve a limit of detection (LOD) as low as 6.6 aM with excellent selectivity. As far as we know, this is the first digital isothermal platform for quantitative analysis of low-abundance circRNA, which could be applied in liquid biopsy for early diagnosis and prognosis. This novel strategy paves new avenues for the development of the digital platform for accurate quantification of circRNA, to further promote this cutting-edge research area.