Background

Circular RNA (circRNA) is an intriguing class of non-coding RNA that is formed through a unique mechanism known as backsplicing, in which the 5′ and 3′ termini are covalently joined (Jeck and Sharpless, 2014). Due to the absence of free termini in the circular structure, circRNA can easily escape from hydrolysis by numerous cellular exonucleases. CircRNAs can be derived from exons, introns, or both, with a great diversity in length. Emerging evidence has shown that circRNAs are involved in many different biological processes and human diseases by functioning as miRNA sponges, RNA-binding protein sponges, transcriptional regulators, mRNA traps, regulators of assembly and transport of cellular proteins, as well as templates for translation.

Several computational methods, such as CIRI2, CIRCexplorer2, and find_circ, have been developed to identify circRNA by employing alignment-based strategies to recognize the back-spliced junction (BSJ), which is a unique feature of circRNAs (Szabo and Salzman, 2016; Cai et al., 2020). To confirm the presence of a circRNA identified in silico, an experimental approach is applied to validate the computationally predicted circRNAs (Zhang et al., 2016).

Reverse-transcription PCR (RT-PCR) is commonly used for the validation of circRNAs identified by RNA sequencing using divergent primers. In this approach, circRNA is converted to cDNA via reverse transcription (RT) with the presence of random hexamer or gene-specific reverse primers. Priming with random hexamers offers flexibility by ensuring RT of all RNA sequences, but gene-specific RT enhances the sensitivity of detecting the target of interest by converting the target of interest—instead of transcribing all RNA in the mix—into cDNA. Priming with gene-specific primers could enhance the detection of circRNA of interest, especially for those present in low abundance (Bustin and Nolan, 2004).

The conventional RT-PCR assay using convergent primers is unable to differentiate circRNAs from their linear transcript when the linear genome is used as template for primer design. Divergent primers are needed for the RT-PCR, as they allow direct detection of BSJ and quantification of circRNA. Divergent primers are a pair of outward-facing primers that allow amplification of circRNAs and not linear mRNA with the same sequence in cDNA and not gDNA (Figure 1). Sanger sequencing is usually performed on the PCR product amplified with the divergent primers to confirm the presence of a BSJ, as to rule out any incorrect amplification or artifact. Quantitative RT-PCR (RT-qPCR) can be used to relatively quantify the expression of circRNA across a panel of samples.

Figure 1. Primer design for circular RNA (circRNA) detection. Divergent primers are a pair of outward-facing primers that allow amplification of circRNA backsplice junction (BSJ), whereas convergent primers are a pair of inward-facing primers that allow amplification of linear mRNA from prepared cDNA.

Combining subcellular fractionation and RT-qPCR with divergent primers may provide some hints on circRNA’s biological functions. For example, circRNAs that are predominantly found in the cytoplasm are more likely to be involved in post-transcriptional gene regulation, whereas nuclear-retained circRNAs are predicted to play a role in transcription regulation. Currently available protocols are mainly for protein work, describing the steps for subcellular fractionation followed by western blot analysis, which is not entirely suitable for RNA work. Instead, this protocol allows the downstream detection of circRNA through RT-qPCR with divergent primers. A detailed protocol for the generation of specific circRNAs-enriched cDNA will also be included to better detect low-abundance circRNAs.