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Detection of cytosine methylation in RNA using bisulfite sequencing

热1 tlexander 添加于 2010-11-15 00:46 | 3573 次阅读 | 0 个评论

作者
Pollex T, Hanna K, Schaefer M
摘要
Post-transcriptional RNA modifications are a characteristic feature of noncoding RNAs and have been described for ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), and various other small RNAs. However, the biological function of most of these modifications remains uncharacterized. Cytosine-5 methylation (5mC) has been detected in abundant and long-lived RNA molecules such as rRNAs and tRNAs, but, because of technical limitations, the occurrence of base-methylated cytosines in other RNAs is not known. To facilitate the detection of RNA methylation, we have established a method for analyzing base-methylated cytosines in RNA using bisulfite sequencing. Treatment of RNA with bisulfite causes the chemical deamination of nonmethylated cytosines to uracil, while methylated cytosines remain unaffected. cDNA synthesis followed by polymerase chain reaction (PCR) amplification and DNA sequencing allows investigators to reproducibly and quantitatively distinguish unmethylated cytosines (as thymines) from methylated cytosines in tRNAs and rRNAs. Using high-throughput sequencing approaches, this protocol should enable the characterization of 5mC methylation patterns in any RNA molecule, including low abundance RNAs.
详细资料
- 文献种类: Journal Article
- 期刊名称: Cold Spring Harbor Protocols
- 期刊缩写: Cold Spring Harb Protoc
- 期卷页: 2010年第2010卷 pdb.prot5505页
- 地址: Division of Epigenetics, DKFZ-ZMBH Alliance, German Cancer Research Center, Heidelberg 69120, Germany
- ISBN: 1559-6095
- 备注:PMID:20889702
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