Abstract
ABSTRACTDiagnostics 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.
Highlights
Circular RNAs are a newly discovered type of endogenous noncoding RNAs [1] characterized by a closed continuous loop formed by a covalent joint of the downstream 3' end and the upstream 5' end
We verified the discrepancy in the circular RNA (circRNA) quantification by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and reverse transcription-droplet digital polymerase chain reaction (RT-droplet digital PCR (ddPCR)), which is caused by the rolling reverse transcription (RT) product originating from the circular structure
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
Summary
Circular RNAs (circRNAs) are a newly discovered type of endogenous noncoding RNAs [1] characterized by a closed continuous loop formed by a covalent joint of the downstream 3' end and the upstream 5' end. The closed loop structure without 5 0–3 0 polarity or polyadenylated tail renderscircRNA remarkably stable and insusceptible to degradation by RNA exonuclease or Ribonuclease R (RNase R) compared with mRNA [2]. Statistically based splicing detection revealed that tissue-specific RNAs in the heart and lung are markedly induced during human fetal development [3]. Recent studies in this field have revealed that circRNAsare involved in several diseases, such as Alzheimer's disease [4], heart failure [9], Moyamoy disease [10], and cancers [5,6,11,12,13,14], whereby they function as miRNA sponges
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