Haematology and Genetic Pathology, Flinders University and Medical Centre, Bedford Park, Adelaide, SA Australia.
Epigenetics. 2012 Jan 1;7(1):92-105. doi: 10.4161/epi.7.1.18815.
We present here the first high resolution melt (HRM) assay to quantitatively analyze differences in murine DNA methylation levels utilizing CpG methylation of Long Interspersed Elements-1 (LINE1 or L1). By calculating the integral difference in melt temperature between samples and a methylated control, and biasing PCR primers for unmethylated CpGs, the assay demonstrates enhanced sensitivity to detect changes in methylation in a cell line treated with low doses of 5-aza-2'-deoxycytidine (5-aza). The L1 assay was confirmed to be a good marker of changes in DNA methylation of L1 elements at multiple regions across the genome when compared with total 5-methyl-cytosine content, measured by Liquid Chromatography-Mass Spectrometry (LC-MS). The assay design was also used to detect changes in methylation at other murine repeat elements (B1 and Intracisternal-A-particle Long-terminal Repeat elements). Pyrosequencing analysis revealed that L1 methylation changes were non-uniform across the CpGs within the L1-HRM target region, demonstrating that the L1 assay can detect small changes in CpG methylation among a large pool of heterogeneously methylated DNA templates. Application of the assay to various tissues from Balb/c and CBA mice, including previously unreported peripheral blood (PB), revealed a tissue hierarchy (from hypermethylated to hypomethylated) of PB > kidney > liver > prostate > spleen. CBA mice demonstrated overall greater methylation than Balb/c mice, and male mice demonstrated higher tissue methylation compared with female mice in both strains. Changes in DNA methylation have been reported to be an early and fundamental event in the pathogenesis of many human diseases, including cancer. Mouse studies designed to identify modulators of DNA methylation, the critical doses, relevant time points and the tissues affected are limited by the low throughput nature and exorbitant cost of many DNA methylation assays. The L1 assay provides a high throughput, inexpensive and sensitive screening tool for identifying and characterizing DNA methylation changes to L1 elements at multiple regions across the genome.
我们在此介绍了第一个高分辨率熔解（HRM）分析，该分析利用长散布元件-1（LINE1 或 L1）的 CpG 甲基化来定量分析小鼠 DNA 甲基化水平的差异。通过计算样品与甲基化对照之间的熔解温度积分差异，并使未甲基化 CpG 的 PCR 引物发生偏置，该分析在经低剂量 5-氮杂-2'-脱氧胞苷（5-aza）处理的细胞系中检测到甲基化变化时具有更高的灵敏度。与通过液相色谱-质谱（LC-MS）测量的基因组多个区域的 L1 元件的总 5-甲基胞嘧啶含量相比，L1 分析被证实是 L1 元件 DNA 甲基化变化的良好标志物。该分析设计还用于检测其他小鼠重复元件（B1 和核内 A-颗粒长末端重复元件）的甲基化变化。焦磷酸测序分析显示，L1 甲基化变化在 L1-HRM 靶区内的 CpG 内不均匀，表明 L1 分析可以检测到大量异质甲基化 DNA 模板中 CpG 甲基化的微小变化。该分析应用于包括先前未报道的外周血（PB）在内的 Balb/c 和 CBA 小鼠的各种组织中，揭示了一种组织层次结构（从高甲基化到低甲基化），即 PB>肾>肝>前列腺>脾。CBA 小鼠的整体甲基化水平高于 Balb/c 小鼠，并且在两种品系中，雄性小鼠的组织甲基化水平均高于雌性小鼠。DNA 甲基化的变化已被报道是许多人类疾病（包括癌症）发病机制中的早期和基本事件。旨在鉴定 DNA 甲基化调节剂、关键剂量、相关时间点和受影响组织的小鼠研究受到许多 DNA 甲基化分析方法通量低和成本高的限制。L1 分析提供了一种高通量、廉价和敏感的筛选工具，用于鉴定和表征基因组多个区域的 L1 元件的 DNA 甲基化变化。
