Journal
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
Volume 20, Issue 16, Pages -Publisher
MDPI
DOI: 10.3390/ijms20163988
Keywords
circRNAs; backsplice sequence; divergent primers; RNase R; RT-PCR; alternative splicing; splice variants
Funding
- Institute of Life Sciences, Department of Biotechnology, India
- University Grant Commission, India
- Science & Engineering Research Board, Department of Science & Technology (DST), India [SB/S2/RJN-099/2017]
- National Institute on Aging Intramural Research Program, National Institutes of Health, USA
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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.
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