Pollex Tim, Hanna Katharina, Schaefer Matthias
Division of Epigenetics, DKFZ-ZMBH Alliance, German Cancer Research Center, Heidelberg 69120, Germany.
Cold Spring Harb Protoc. 2010 Oct 1;2010(10):pdb.prot5505. doi: 10.1101/pdb.prot5505.
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.
转录后RNA修饰是非编码RNA的一个特征，并且已在核糖体RNA（rRNA）、转运RNA（tRNA）以及各种其他小RNA中被描述。然而，这些修饰中的大多数的生物学功能仍未得到表征。胞嘧啶-5甲基化（5mC）已在诸如rRNA和tRNA等丰富且长寿的RNA分子中被检测到，但是由于技术限制，其他RNA中碱基甲基化胞嘧啶的存在情况尚不清楚。为了便于检测RNA甲基化，我们建立了一种使用亚硫酸氢盐测序分析RNA中碱基甲基化胞嘧啶的方法。用亚硫酸氢盐处理RNA会使未甲基化的胞嘧啶化学脱氨基变成尿嘧啶，而甲基化的胞嘧啶则不受影响。随后进行聚合酶链反应（PCR）扩增和DNA测序的cDNA合成使研究人员能够在tRNA和rRNA中可重复且定量地将未甲基化胞嘧啶（作为胸腺嘧啶）与甲基化胞嘧啶区分开来。使用高通量测序方法，该方案应该能够表征任何RNA分子中的5mC甲基化模式，包括低丰度RNA。
