Hundreds of thousands of putative circular RNAs have been identified through deep sequencing and bioinformatic analyses. However, the circularity of these putative RNA circles has not been experimentally validated due to limited methodologies currently available. We reported here that the template-switching capability of commonly used reverse transcriptases (e.g., SuperScript II) leads to the formation of artificial junction sequences, and consequently misclassification of large linear RNAs as RNA circles. Use of reverse transcriptases without terminal transferase activity (e.g., MonsterScript) for cDNA synthesis is critical for the identification of physiological circular RNAs. We also report two methods, MonsterScript junction PCR and high-resolution melting curve analyses, which can reliably distinguish circular RNAs from their linear forms and thus, can be used to discover and validate true circular RNAs.
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