Format

Send to

Choose Destination
See comment in PubMed Commons below
Methods Mol Biol. 2004;265:357-91.

Detection and quantification of modified nucleotides in RNA using thin-layer chromatography.

Author information

1
Laboratoire d'Enzymolgie et Biochimie Structurales du CNRS, Gif-sur-Yvette, France.

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 two-dimensional 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.

PMID:
15103084
DOI:
10.1385/1-59259-775-0:357
[Indexed for MEDLINE]
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for Springer
    Loading ...
    Support Center