RNA 5-甲基胞嘧啶的定量和单核苷酸分辨率分析。

Quantitative and Single-Nucleotide Resolution Profiling of RNA 5-Methylcytosine.

机构信息

Department of Molecular and Biomedical Sciences, School of Biological Sciences, The University of Adelaide, Adelaide, SA, Australia.

出版信息

Methods Mol Biol. 2021;2298:135-151. doi: 10.1007/978-1-0716-1374-0_9.

DOI:10.1007/978-1-0716-1374-0_9

PMID:34085243

Abstract

RNA has coevolved with numerous posttranscriptional modifications to sculpt interactions with proteins and other molecules. One of these modifications is 5-methylcytosine (mC) and mapping the position and quantifying the level in different types of cellular RNAs and tissues is an important objective in the field of epitranscriptomics. Both in plants and animals bisulfite conversion has long been the gold standard for detection of mC in DNA but it can also be applied to RNA. Here, we detail methods for highly reproducible bisulfite treatment of RNA, efficient locus-specific PCR amplification, detection of candidate sites by sequencing on the Illumina MiSeq platform, and bioinformatic calling of non-converted sites.

摘要

RNA 与众多转录后修饰共同进化，以塑造与蛋白质和其他分子的相互作用。这些修饰之一是 5-甲基胞嘧啶（mC），在不同类型的细胞 RNA 和组织中定位和定量其水平是表观转录组学领域的一个重要目标。在植物和动物中，亚硫酸氢盐转化长期以来一直是检测 DNA 中 mC 的金标准，但它也可以应用于 RNA。在这里，我们详细介绍了高度可重复的 RNA 亚硫酸氢盐处理、高效的特定基因座 PCR 扩增、在 Illumina MiSeq 平台上测序检测候选位点，以及非转化位点的生物信息学调用方法。