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Identification of Circular RNAs using RNA Sequencing
Published on: November 14, 2019
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Validation of Circular RNAs by PCR
Aniruddha Das1,2, Debojyoti Das1,2, Amaresh C Panda3
1Institute of Life Sciences, Bhubaneswar, Odisha, India.
Methods in Molecular Biology (Clifton, N.J.)
|November 13, 2021
Summary
Circular RNAs (circRNAs) are a newly discovered class of RNA molecules. This guide provides essential primer design strategies for validating circRNAs using reverse transcription polymerase chain reaction (RT-PCR).
Area of Science:
- Molecular Biology
- Genomics
- Bioinformatics
Background:
- High-throughput RNA-sequencing (RNA-seq) has identified millions of circular RNAs (circRNAs).
- Experimental validation of circRNAs remains crucial, with limited methods currently employed.
- Reverse transcription polymerase chain reaction (RT-PCR) is a widely adopted technique for circRNA detection.
Purpose of the Study:
- To provide guidelines for designing primers for circRNA validation.
- To detail the application of RT-PCR for circRNA detection and quantification.
Main Methods:
- Utilizing RNA-sequencing data to identify potential circRNAs.
- Employing polymerase chain reaction (PCR) with specific primer design for circRNA amplification.
- Implementing reverse transcription (RT) of total RNA prior to PCR.
Main Results:
- RT-PCR offers a sensitive, reproducible, and quantitative method for circRNA validation.
- Successful primer design is critical for accurate circRNA detection via RT-PCR.
Conclusions:
- Effective primer design is fundamental for validating circRNAs identified by RNA-seq.
- RT-PCR is a robust method for the detection and quantification of circular RNAs.
