Beckman Coulter Biomedical GmbH, Advalytix Products, Munich, Germany.
Nucleic Acids Res. 2011 Apr;39(7):e44. doi: 10.1093/nar/gkq1357. Epub 2011 Jan 25.
In recent years, the field of epigenetics has grown dramatically and has become one of the most dynamic and fast-growing branches of molecular biology. The amount of diseases suspected of being influenced by DNA methylation is rising steadily and includes common diseases such as schizophrenia, bipolar disorder, Alzheimer's disease, diabetes, atherosclerosis, cancer, major psychosis, lupus and Parkinson's disease. Due to cellular heterogeneity of methylation patterns, epigenetic analyses of single cells become a necessity. One rationale is that DNA methylation profiles are highly variable across individual cells, even in the same organ, dependent on the function of the gene, disease state, exposure to environmental factors (e.g. radiation, drugs or nutrition), stochastic fluctuations and various other causes. Using a polymerase chain reaction (PCR)-slide microreaction system, we present here a methylation-sensitive PCR analysis, the restriction enzyme-based single-cell methylation assay (RSMA), in the analysis of DNA methylation patterns in single cells. This method addresses the problems of cell heterogeneity in epigenetics research; it is comparably affordable, avoids complicated microfluidic systems and offers the opportunity for high-throughput screening, as many single cells can be screened in parallel. In addition to this study, critical principles and caveats of single cell methylation analyses are discussed.
近年来，表观遗传学领域发展迅速，已成为分子生物学中最具活力和发展最快的分支之一。越来越多的疾病被怀疑受到 DNA 甲基化的影响，包括精神分裂症、双相情感障碍、阿尔茨海默病、糖尿病、动脉粥样硬化、癌症、主要精神病、狼疮和帕金森病等常见疾病。由于甲基化模式的细胞异质性，单细胞的表观遗传学分析成为必要。一个理由是，即使在同一器官中，DNA 甲基化图谱在个体细胞之间也高度可变，这取决于基因的功能、疾病状态、暴露于环境因素（例如辐射、药物或营养）、随机波动和各种其他原因。我们在这里使用聚合酶链反应 (PCR)-载玻片微反应系统，提出了一种用于单细胞 DNA 甲基化模式分析的甲基化敏感 PCR 分析方法，即基于限制性内切酶的单细胞甲基化分析 (RSMA)。该方法解决了表观遗传学研究中细胞异质性的问题；它的成本相对较低，避免了复杂的微流控系统，并提供了高通量筛选的机会，因为可以并行筛选许多单细胞。除了这项研究外，还讨论了单细胞甲基化分析的关键原则和注意事项。
