Department of Molecular Biology and Genetics, Faculty of Science, Karadeniz Technical University, 61080, Trabzon, Turkey.
Anal Biochem. 2020 Jul 15;601:113789. doi: 10.1016/j.ab.2020.113789. Epub 2020 May 27.
Cytosine methylation is the leading epigenetic modification on DNA playing a role in gene regulation. Methylation can occur in cytosines of any nucleic acids in cytosol (as mitochondrial DNA, mtDNA) and in nuclear DNA (ncDNA). mtDNA exists as multiple copies within numerous mitochondria. This suggests that the number of mitochondria and mtDNA copy number can indicate the presence of a significant amount of DNA methylation within total DNA methylation detected. However, immunofluorescence method does not have a step to discriminate the staining between ncDNA and mtDNA. Antibodies used in immunological methods are methylation-specific but not selective for DNA type and they can bind to methylated cytosines in any DNA within the specimen. Current study aimed to understand whether mtDNA methylation interferes with the detection of nuclear DNA methylation by immunofluorescence and affinity enrichment (ELISA) in different mammalian cells. Experiments were performed to distinguish methylation between mtDNA and ncDNA. Immunofluorescence showed that there was no significant difference in the detected amount of methylation between mitochondrial and nuclear DNA. But ELISA revealed that up to 25% of cellular methylation was derived from mitochondria. This suggests that significant contamination of mtDNA methylation with ncDNA methylation can result in overestimation of the quantitative level of nuclear methylation.
胞嘧啶甲基化是 DNA 上主要的表观遗传修饰，在基因调控中发挥作用。甲基化可以发生在细胞质中任何核酸的胞嘧啶上（如线粒体 DNA，mtDNA）和核 DNA（ncDNA）上。mtDNA 以多个拷贝存在于众多线粒体中。这表明线粒体的数量和 mtDNA 拷贝数可以表明在检测到的总 DNA 甲基化中存在大量的 DNA 甲基化。然而，免疫荧光法没有区分 ncDNA 和 mtDNA 染色的步骤。免疫学法中使用的抗体是甲基化特异性的，但不是针对 DNA 类型的选择性抗体，它们可以与标本中任何 DNA 中的甲基化胞嘧啶结合。本研究旨在了解 mtDNA 甲基化是否会干扰不同哺乳动物细胞中免疫荧光和亲和富集（ELISA）检测核 DNA 甲基化。进行了实验以区分 mtDNA 和 ncDNA 之间的甲基化。免疫荧光显示线粒体和核 DNA 中检测到的甲基化量没有显著差异。但 ELISA 显示，高达 25%的细胞甲基化来自线粒体。这表明 mtDNA 甲基化与 ncDNA 甲基化的严重污染会导致核甲基化定量水平的高估。
