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

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Plant Circular RNAs

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2362))

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.

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Acknowledgments

We acknowledge the fund granted from the Science and Engineering Research Board (SERB) (Ref. No. EEQ/2018/000067, SB/EMEQ-070/2013 to GP and EMR/2016/000945 to SN). PS is a recipient of Lady Tata Memorial Trust (LTMT) Junior Research Scholarship (2019-2020). Financial assistance was provided to AG by Department of Biotechnology (DBT) (Ref. No. BT/PR23641/BPA/118/309/2017, BT/PR2061/AGR/36/707/2011) and grants from BT/PR6466/COE/34/16/2012. The equipment grants from Department of Science and Technology—Promotion of University Research and Scientific Excellence (DST-PURSE), University Grant Commission—Special Assistance Programme (UGC-SAP) are gratefully acknowledged.

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Author notes

  1. Ashirbad Guria, Priyanka Sharma contributed equally to this work.

Authors and Affiliations

  1. Department of Plant Biotechnology, School of Biotechnology, Madurai Kamaraj University, Madurai, India

    Ashirbad Guria & Gopal Pandi

  2. Department of Genetic Engineering, School of Biotechnology, Madurai Kamaraj University, Madurai, India

    Priyanka Sharma & Sankar Natesan

Authors
  1. Ashirbad Guria
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  2. Priyanka Sharma
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  3. Sankar Natesan
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  4. Gopal Pandi
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Corresponding authors

Correspondence to Sankar Natesan or Gopal Pandi .

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Editors and Affiliations

  1. Oncativo, Argentina

    Luis María Vaschetto

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© 2021 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Guria, A., Sharma, P., Natesan, S., Pandi, G. (2021). Identification of Circular RNAs by Multiple Displacement Amplification and Their Involvement in Plant Development. In: Vaschetto, L.M. (eds) Plant Circular RNAs. Methods in Molecular Biology, vol 2362. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1645-1_4

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  • DOI: https://doi.org/10.1007/978-1-0716-1645-1_4

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1644-4

  • Online ISBN: 978-1-0716-1645-1

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