Circular RNA profiling by Illumina sequencing via template-dependent multiple displacement amplification

Circular RNAs (circRNAs) are newly discovered incipient non-coding RNAs with potential roles in disease progression in living organisms. Significant reports, since their inception, highlight the abundance and putative functional roles of circRNAs in every organism checked for, like O. sativa, Arabidopsis, human, and mouse. CircRNA expression is generally less than their linear mRNA counterparts which fairly explains the competitive edge of canonical splicing over non-canonical splicing. However, existing methods may not be sensitive enough for the discovery of low-level expressed circRNAs.

By combining template-dependent multiple displacement amplification (tdMDA), Illumina sequencing, and bioinformatics tools, researchers from Madurai Kamaraj University and  Saint Louis University have developed an experimental protocol that is able to detect 1,875 novel and known circRNAs from O. sativa. The same method also revealed 9,242 putative circRNAs in less than 40 million reads for the first time from the Nicotiana benthamiana whose genome has not been fully annotated. Supported by the PCR-based validation and Sanger sequencing of selective circRNAs, this method represents a valuable tool in profiling circRNAs from the organisms with or without genome annotation.

Divergent PCR for validation of NGS-tdMDA derived circRNA

Two products (>100bp and >200bp) were amplified with nb_circ7 primer upon divergent PCR (lane 4,5,6) with three N. benthamiana cDNA (a). With same primer, it did not give same size amplicon with genomic DNA (lane 1,2,3) as template (b). Two amplicons at ~100bp and 270 bp formed from rice cDNA (lane3) with osi_circ10 divergent primer whereas non-specific amplicon also formed when taking genomic DNA as template (lane 2) (c). Non-template PCR was taken as negative control (lane 2 in (a), lane 5 in (b), and lane 4 in (c)) and 100 bp amplicon formed from 5.8s as positive control (lane 1 in (a), lane 4 in (b), and lane 5 in (c)). Generuler 100 bp plus ladder in lane 3 (a), lane 6 (b), and Fermentas 100 bp ladder in lane 1 (c).