Table 3 Methods of circRNA detection
Method | Principle | Purpose | Ref. |
|---|---|---|---|
Bioinformatics | The online database, such as circBase, Circular RNA Interactome, miRBase, DIANA TOOLS, and Cytoscape | Predicting circRNA interactive network and molecular mechanism | |
qRT-PCR | Primers were designed for the back-splicing junction to detect circRNA expression quantitatively | Detecting circRNA expression | [19] |
ddPCR | Primers were designed for the back-splicing junction to detect circRNA expression quantitatively | Detecting circRNA expression | [97] |
RNase R treatment | RNase R is a magnesium-dependent 3′ → 5′ exoribonuclease, owning that circRNA or lariat lack this structure so that they can resist its digestion | Verifying the structure of circRNA | [1] |
Northern blotting | Probes were designed for the back-splicing junction to detect circRNA expression quantitatively | Detecting circRNA expression | [98] |
FISH | Fluorescent probes were designed for circRNA and the miRNA binding sites | Analyzing co-location of circRNA and miRNA by in situ imaging | [98] |
RCA reaction | CircRNA hybridizes with miRNA to form complex cmRRIs, which trigger RCA reactions. Probes are designed for rolling ring amplification products to detect fluorescence intensity | Detecting circRNA expression and judging the affinity of circRNA to miRNA | |
RNA sequencing | The sequence of circRNA was analyzed by high-throughput sequencing | Studying genome-wide differences of circRNAs | |
Microarray | CircRNA microarray, which is not affected by RNA abundance, can accurately detect the expression of circRNA in samples by using the double guarantee of a specific splicing site probe and exonuclase pretreatment | Microarray allows fastly, primarily, and more sensitively screen circRNA. | [101] |
CRISPR-Cas13 system | CRISPR-RfxCas13d can effectively discriminate circRNAs from mRNAs by using guide RNAs targeting sequences spanning back-splicing junction sites featured in RNA circles | CRISPR-RfxCas13d is a useful tool for the discovery and functional study of circRNAs at both individual and large-scale levels | [96] |