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

  • Henri Grosjean
  • Gérard Keith
  • Louis Droogmans
Part of the Methods in Molecular Biology book series (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.

Key Words

RNA maturation posttranscriptional modification thin-layer chromatography sequencing modified nucleotides 2′-O-methylation nuclease RNase kinase phosphatase 

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© Humana Press Inc. 2004

Authors and Affiliations

  • Henri Grosjean
    • 1
  • Gérard Keith
    • 2
  • Louis Droogmans
    • 3
  1. 1.Laboratoire d’Enzymologie et Biochimie Structurales du CNRSGif-sur-YvetteFrance
  2. 2.Institut de Biologie Moléculaire et Cellulaire du CNRSStrasbourg CedexFrance
  3. 3.Laboratoire de Microbiologie Institut de Recherches Microbiologiques J. M. WiameUniversité Libre de BruxellesBruxellesBelgium

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