Abstract
High-throughput RNA-sequencing (RNA-seq) technologies combined with novel bioinformatic algorithms discovered a large class of covalently closed single-stranded RNA molecules called circular RNAs (circRNAs ). Although RNA-seq has identified more than a million circRNAs, only a handful of them is validated with other techniques, including northern blotting, gel-trap electrophoresis, exonuclease treatment assays, and polymerase chain reaction (PCR). Reverse transcription (RT) of total RNA followed by PCR amplification is the most widely used technique for validating circRNAs identified in RNA-seq. RT-PCR is a highly reproducible, sensitive, and quantitative method for the detection and quantitation of circRNAs. This chapter details the basic guidelines for designing suitable primers for PCR amplification and validation of circRNAs .
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Acknowledgments
This work was supported by the DBT/Wellcome Trust India Alliance Fellowship [grant number IA/I/18/2/504017] awarded to Amaresh Panda and intramural support from Institute of Life Sciences, DBT, India. Aniruddha Das and Debojyoti Das are supported by University Grant Commission of India.
Conflicts of interest: The authors declare no conflict of interest.
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Das, A., Das, D., Panda, A.C. (2022). Validation of Circular RNAs by PCR. In: Basu, C. (eds) PCR Primer Design. Methods in Molecular Biology, vol 2392. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1799-1_8
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DOI: https://doi.org/10.1007/978-1-0716-1799-1_8
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