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Nucleic Acids Research Advance Access published online on December 5, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn954
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© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

RNA cytosine methylation analysis by bisulfite sequencing

Matthias Schaefer*, Tim Pollex, Katharina Hanna and Frank Lyko

Division of Epigenetics, German Cancer Research Center, Heidelberg, Germany

*To whom correspondence should be addressed. Tel: +49 6221 423804; Fax: +49 6221 423802; Email: m.schaefer{at}dkfz.de

Received September 24, 2008. Revised November 7, 2008. Accepted November 11, 2008.

Covalent modifications of nucleic acids play an important role in regulating their functions. Among these modifications, (cytosine-5) DNA methylation is best known for its role in the epigenetic regulation of gene expression. Post-transcriptional RNA modification is a characteristic feature of noncoding RNAs, and has been described for rRNAs, tRNAs and miRNAs. (Cytosine-5) RNA methylation has been detected in stable and long-lived RNA molecules, but its function is still unclear, mainly due to technical limitations. In order to facilitate the analysis of RNA methylation patterns we have established a protocol for the chemical deamination of cytosines in RNA, followed by PCR-based amplification of cDNA and DNA sequencing. Using tRNAs and rRNAs as examples we show that cytosine methylation can be reproducibly and quantitatively detected by bisulfite sequencing. The combination of this method with deep sequencing allowed the analysis of a large number of RNA molecules. These results establish a versatile method for the identification and characterization of RNA methylation patterns, which will be useful for defining the biological function of RNA methylation.


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