Masiello Irene, Biggiogera Marco
Laboratory of Cell Biology and Neurobiology, Department of Biology and Biotechnology, University of Pavia, Pavia, Italy.
Methods Mol Biol. 2019;1870:165-177. doi: 10.1007/978-1-4939-8808-2_12.
5-Methylcytosine is the major epigenetic modification occurring on DNA. It is known to be involved not only in gene expression regulation but also in the control of chromatin structure. However, this modification is also found on different types of RNA, including mRNA. Generally, biomolecular techniques are applied for studying the epigenetic profile of nucleic acids. Here, we describe the ultrastructural detection of 5-methylcytosine as an unusual approach to localize this modification on chromatin regions and/or RNA single molecules. This tool requires a careful sample preparation to preserve antigen epitopes that will be revealed immunocytochemically by a specific anti-5-methylcytosine antibody. The multiple staining procedures that can be adopted allow the identification of both DNA or RNA. A semiquantitative analysis can also be carried out.
5-甲基胞嘧啶是DNA上发生的主要表观遗传修饰。已知它不仅参与基因表达调控，还参与染色质结构的控制。然而，这种修饰也存在于不同类型的RNA上，包括信使核糖核酸（mRNA）。一般来说，生物分子技术被应用于研究核酸的表观遗传特征。在这里，我们描述了5-甲基胞嘧啶的超微结构检测，这是一种在染色质区域和/或RNA单分子上定位这种修饰的独特方法。该工具需要精心的样品制备，以保留抗原表位，这些表位将通过特异性抗5-甲基胞嘧啶抗体进行免疫细胞化学检测。可以采用的多重染色程序能够识别DNA或RNA。还可以进行半定量分析。
