Virginia Tech-Wake Forest University School of Biomedical Engineering and Sciences, Wake Forest School of Medicine, Winston-Salem, NC, USA.
Department of Engineering, Wake Forest University, Winston-Salem, NC, USA.
Nanomedicine. 2021 Jul;35:102407. doi: 10.1016/j.nano.2021.102407. Epub 2021 Apr 24.
5-Hydroxymethylcytosine (5hmC), the first oxidized form of the well-known epigenetic modification 5-methylcytosine, is an independent regulator of gene expression and therefore a potential marker for disease. Here, we report on methods developed for a selective solid-state nanopore assay that enable direct analysis of global 5hmC content in human tissue. We first describe protocols for preparing genomic DNA derived from both healthy breast tissue and stage 1 breast tumor tissue and then use our approach to probe the net abundance of the modified base in each cohort. Then, we employ empirical data to adjust for the impact of nanopore diameter on the quantification. Correcting for variations in nanopore diameter among the devices used for analysis reveals no detectable difference in global 5hmC content between healthy and tumor tissue. These results suggest that 5hmC changes may not be associated with early-stage breast cancer and instead are a downstream consequence of the disease.
5-羟甲基胞嘧啶（5hmC）是广为人知的表观遗传修饰 5-甲基胞嘧啶的第一种氧化形式，是基因表达的独立调控因子，因此可能成为疾病的潜在标志物。在这里，我们报告了开发用于选择性固态纳米孔分析的方法，该方法可直接分析人类组织中的全局 5hmC 含量。我们首先描述了从健康乳腺组织和 1 期乳腺癌组织中提取基因组 DNA 的方案，然后使用我们的方法来探测每个队列中修饰碱基的净丰度。然后，我们利用经验数据来调整纳米孔直径对定量的影响。校正分析中使用的设备之间的纳米孔直径变化，在健康组织和肿瘤组织之间未发现全局 5hmC 含量的可检测差异。这些结果表明，5hmC 的变化可能与早期乳腺癌无关，而是疾病的下游结果。
