Identification of Circular RNAs by Multiple Displacement Amplification and Their Involvement in Plant Development.

Abstract

With the innovative knowledge and bioinformatics tools in the identification and characterization of noncoding RNAs, circular RNA (circRNA) is added as a new member to the noncoding RNAs family. CircRNA enrichment by rRNA depletion/RNase R or poly-A removal/RNase R treatment followed by NGS analysis is the most frequently adopted method for circular RNA identification and characterization. In this chapter, we describe the multiple displacement amplification (MDA) as a convenient method to augment the identification of even the abysmally expressed circular RNAs at low sequencing depth. Total RNA, extracted at three different developmental stages of rice, is subjected to RiboMinus and RNase R treatment to deplete the linear RNAs. The enriched circular RNAs are reverse transcribed with random hexamers. The resulting cDNA is subjected to phi29 DNA polymerase amplification using exo-resistant random pentamers to yield high molecular weight dsDNA product, followed by Illumina sequencing at ten million paired end reads per sample. The sequence analysis yielded a promising number of circRNAs with the appreciable inclusion of differentially regulated and minimally expressed circRNAs at a comparatively reduced cost.