Application of droplet digital PCR in quantitative detection of the cell-free circulating circRNAs

Diagnostics based on circulating circular RNA (circRNA) is an emerging field for noninvasive molecular diagnosis, owing to the stable circular structure of circRNA. However, the uniquely circular structure still poses a challenge for circRNA quantification in the research community. Here, we verified the discrepancy in the circRNA quantification by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and reverse transcription-droplet digital polymerase chain reaction (RT-ddPCR), which is caused by the rolling reverse transcription (RT) product originating from the circular structure. In addition, we detected the stability of cell-free circRNA in serum/plasma and determined the pre-analysis sampling procedure that will compromise the quantification of circRNA. Our results showed that prolonged RT incubation time resulted in multiple PCR products from circular RNA, which will reduce the accuracy of circRNA quantification by RT-qPCR technology, whereas ddPCR can address this limitation and could be a good alternative to qPCR for circRNA quantification. CircRNA showed a high stability in promptly separated serum/plasma but not in delay-separated samples.