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Rolling Circle cDNA Synthesis Uncovers Circular RNA Splice Variants

Institute of Life Sciences, Nalco Square, Bhubaneswar 751023, India
School of Biotechnology, KIIT University, Bhubaneswar 751023, India
Laboratory of Genetics and Genomics, National Institute on Aging, NIH, Baltimore, MD 21224, USA
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2019, 20(16), 3988;
Received: 9 July 2019 / Revised: 29 July 2019 / Accepted: 31 July 2019 / Published: 16 August 2019
(This article belongs to the Collection Regulation by Non-Coding RNAs)
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High-throughput RNA sequencing and novel bioinformatic pipelines have identified thousands of circular (circ)RNAs containing backsplice junction sequences. However, circRNAs generated from multiple exons may contain different combinations of exons and/or introns arising from alternative splicing, while the backsplice junction sequence is the same. To be able to identify circRNA splice variants, we developed a method termed circRNA-Rolling Circle Amplification (circRNA-RCA). This method detects full-length circRNA sequences by performing reverse transcription (RT) in the absence of RNase H activity, followed by polymerase chain reaction (PCR) amplification of full-length circRNAs using a forward primer spanning the backsplice junction sequence and a reverse primer exactly upstream of the forward primer. By sequencing the PCR products, circRNA splice variants bearing the same backsplice junctions, which were otherwise only predicted computationally, could be experimentally validated. The splice variants were further predicted to associate with different subsets of target RNA-binding proteins and microRNAs, supporting the notion that different circRNA splice variants can have different biological impacts. In sum, the circRNA-RCA method allows the accurate identification of full-length circRNA sequences, offering unique insight into their individual function. View Full-Text
Keywords: circRNAs; backsplice sequence; divergent primers; RNase R; RT-PCR; alternative splicing; splice variants circRNAs; backsplice sequence; divergent primers; RNase R; RT-PCR; alternative splicing; splice variants

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Das, A.; Rout, P.K.; Gorospe, M.; Panda, A.C. Rolling Circle cDNA Synthesis Uncovers Circular RNA Splice Variants. Int. J. Mol. Sci. 2019, 20, 3988.

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