Circular RNA Profiling by Illumina Sequencing via Template-Dependent Multiple Displacement Amplification

Area of Science:

• Molecular Biology
• Genomics
• Plant Science

Background:

• Circular RNAs (circRNAs) are emerging non-coding RNAs with significant roles in various biological processes and diseases.
• While abundant, circRNAs are often expressed at lower levels than linear mRNAs, posing challenges for detection.
• Existing methods may lack the sensitivity to identify low-abundance circRNAs.

Purpose of the Study:

• To develop a highly sensitive experimental protocol for detecting and profiling circular RNAs (circRNAs).
• To identify novel and known circRNAs in organisms with and without fully annotated genomes.

Main Methods:

• Combined template-dependent multiple displacement amplification (tdMDA) with Illumina sequencing.
• Utilized bioinformatics tools for data analysis.
• Validated findings using PCR and Sanger sequencing.

Main Results:

• Identified 1,875 novel and known circRNAs in O. sativa (rice).
• Discovered 9,242 putative circRNAs in Nicotiana benthamiana (tobacco), a first for this species.
• Demonstrated the method's effectiveness for organisms with or without genome annotation.

Conclusions:

• The developed protocol significantly enhances the detection of circRNAs, including low-abundance ones.
• This method is a valuable tool for circRNA profiling across diverse species.
• Facilitates circRNA research in both model and non-model organisms.