Ghantous Akram, Hernandez-Vargas Hector, Herceg Zdenko
Epigenetics Group, International Agency for Research on Cancer (IARC), 150 rue Albert-Thomas, 69008, Lyon, France.
Methods Mol Biol. 2018;1708:605-619. doi: 10.1007/978-1-4939-7481-8_31.
Blood represents an easily accessible human tissue for numerous research and clinical applications, including surrogate roles for biomarkers of other tissues. Dried blood spots (DBS) are space- and cost-efficient storage forms of blood while stably retaining many of its chemical constituents. Consequently, neonatal DBS are routinely collected in many countries, and their biobanks represent gold mines for research. However, the utility of DBS is restricted by the limited amount and quality of extractable biomolecules (including DNA), especially for genome-wide profiling. In particular, DNA methylome analysis in DBS has proven to be technically challenging, mainly due to the requirement for stringent preprocessing, such as bisulfite conversion. Moreover, DNA amplification, required to increase its yield, often leads to a bias in the analysis, particularly in methylome profiles. Thus, it is important to develop methodologies that maximize both the yield and quality of DNA from DBS for downstream analyses. Using a combination of in-house-derived and modified commercial extraction methods, we developed two robust protocols that produced increased DNA yield and quality from DBS. Though both protocols are more efficient relative to other published methods, one protocol yields less DNA compared to the other, but shows improved 260/280 spectrophotometric ratios, which are useful for sample quality assessment. Both protocols consist of two sequential phases, each involving several critical steps. Phase I comprises blood extraction off the filter papers, cell lysis, and protein digestion. Phase II involves DNA precipitation, purification, and elution. Results from subsequent locus-specific and genome-wide DNA methylation analyses demonstrate the high quality, reproducibility, and consistency of the data. This work may prove useful to meet the increased demand for research on DBS, particularly with a focus on the epigenetic origins of human diseases and newborn screening programs.
血液是一种易于获取的人体组织，可用于众多研究和临床应用，包括作为其他组织生物标志物的替代物。干血斑（DBS）是一种节省空间和成本的血液储存形式，同时能稳定保留其许多化学成分。因此，许多国家都常规采集新生儿干血斑，其生物样本库是研究的宝库。然而，DBS的效用受到可提取生物分子（包括DNA）数量和质量的限制，特别是对于全基因组分析而言。尤其是，DBS中的DNA甲基化组分析已被证明在技术上具有挑战性，主要是因为需要进行严格的预处理，如亚硫酸氢盐转化。此外，为了提高产量而进行的DNA扩增往往会导致分析偏差，特别是在甲基化组图谱中。因此，开发能够最大限度提高DBS中DNA产量和质量以用于下游分析的方法非常重要。通过结合内部衍生和改良的商业提取方法，我们开发了两种强大的方案，可提高DBS中DNA的产量和质量。虽然这两种方案相对于其他已发表的方法都更有效，但其中一种方案产生的DNA比另一种少，但显示出改善的260/280分光光度比，这对样本质量评估很有用。两种方案都包括两个连续阶段，每个阶段都涉及几个关键步骤。第一阶段包括从滤纸上提取血液、细胞裂解和蛋白质消化。第二阶段涉及DNA沉淀、纯化和洗脱。后续位点特异性和全基因组DNA甲基化分析的结果证明了数据的高质量、可重复性和一致性。这项工作可能有助于满足对DBS研究日益增长的需求，特别是关注人类疾病的表观遗传起源和新生儿筛查项目。
