Sanger Sequencing to Determine the Full-Length Sequence of Circular RNAs

The pre-existing theory of pre-mRNA splicing into linear mature RNA was questioned with the introduction of circular RNAs (circRNAs). Hundreds of studies using high throughput RNA-sequencing (RNA-seq) techniques and novel computational programs reported the abundant and ubiquitous expression of circRNAs originating by pre-mRNA backsplicing. CircRNAs are mostly involved in gene expression by regulating functions of interacting microRNAs (miRNAs) and RNA-binding proteins (RBPs) or translating into functional polypeptides. Although all circRNA annotation tools identify circRNAs based on the backsplice junction (BSJ) sequences, only a few identify the internal sequences of circRNAs. However, the full-length sequence of circRNAs from RNA-seq data could be error-prone due to its similarity with the counterpart linear RNA. Since circRNA function depends on the mature sequence, validation of the mature sequence is the prerequisite for their further characterization. In this chapter, we discuss the validation of circRNA BSJ sequence by RT-PCR using divergent primer followed by Sanger sequencing. Furthermore, we describe the circRNA-rolling circle amplification (circRNA-RCA; circRNA enrichment by RNase R treatment, full-length cDNA synthesis, rolling circle PCR amplification using full-length primers, and Sanger sequencing of the PCR product) to validate the mature splice sequence of circRNAs. This chapter highlights the basic guidelines for designing divergent and full-length primers for PCR amplification and Sanger sequencing to validate circRNA sequences.