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RNA Interference, Editing, and Modification pp 357–391Cite as

Detection and Quantification of Modified Nucleotides in RNA Using Thin-Layer Chromatography

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 265))

Abstract

Identification of a modified nucleotide and its localization within an RNA molecule is a difficult task. Only direct sequencing of purified RNA molecules and high-performance liquid chromatography mass spectrometry analysis of purified RNA fragments allow determination of both the type and location of a given modified nucleotide within an RNA of 50–150 nt in length. The objective of this chapter is to describe in detail a few simple procedures that we have found particularly suited for the detection, localization, and quantification of modified nucleotides within an RNA of known sequence. The methods can also be used to reveal the enzymatic activity of a particular RNA-modifying enzyme in vitro or in vivo. The procedures are based on the use of radiolabeled RNA (with [32P], [14C], or [3H]) or [32P]-postlabeled oligonucleotides and twodimensional thin-layer chromatography of labeled nucleotides on cellulose plates. This chapter provides useful maps of the migration characteristics of 70 modified nucleotides on thin-layer cellulose plates.

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Author information

Authors and Affiliations

  1. Laboratoire d’Enzymologie et Biochimie Structurales du CNRS, Gif-sur-Yvette, France

    Henri Grosjean

  2. Institut de Biologie Moléculaire et Cellulaire du CNRS, Strasbourg Cedex, France

    Gérard Keith

  3. Laboratoire de Microbiologie Institut de Recherches Microbiologiques J. M. Wiame, Université Libre de Bruxelles, Bruxelles, Belgium

    Louis Droogmans

Authors
  1. Henri Grosjean
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  2. Gérard Keith
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  3. Louis Droogmans
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Editor information

Editors and Affiliations

  1. Case Western Reserve University School of Medicine, Cleveland, OH

    Jonatha M. Gott

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© 2004 Humana Press Inc.

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Grosjean, H., Keith, G., Droogmans, L. (2004). Detection and Quantification of Modified Nucleotides in RNA Using Thin-Layer Chromatography. In: Gott, J.M. (eds) RNA Interference, Editing, and Modification. Methods in Molecular Biology, vol 265. Humana Press. https://doi.org/10.1385/1-59259-775-0:357

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  • DOI: https://doi.org/10.1385/1-59259-775-0:357

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-242-1

  • Online ISBN: 978-1-59259-775-8

  • eBook Packages: Springer Protocols

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