Mullegama Sureni V, Alberti Michael O, Au Cora, Li Yan, Toy Traci, Tomasian Vanina, Xian Rena R
Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA.
Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
Methods Mol Biol. 2019;1897:359-383. doi: 10.1007/978-1-4939-8935-5_30.
Nucleic acid isolation is often the starting point for all downstream experiments in biomedical research. It is therefore the most crucial step in any molecular technique. DNA and RNA extraction follow protocols with standardized reagents, many of which are available in quality-controlled commercial kits. Irrespective of the protocol, successful extraction of high-quality nucleic acid from biological tissues requires sufficient disruption of the tissue and cellular structures, denaturation of nucleoprotein complexes, inactivation of nucleases, and nucleic acid purification. These steps can be modified based on nucleic acid of interest and biological sample source. This chapter addresses DNA and RNA extraction from a variety of sample and tissue types, including saliva, and formalin-fixed, paraffin-embedded tissues, which are often archived in clinical pathology laboratories. Special considerations and common pitfalls of each protocol will also be discussed, as will nucleic acid quantitation techniques.
核酸分离通常是生物医学研究中所有下游实验的起点。因此，它是任何分子技术中最关键的步骤。DNA和RNA提取遵循使用标准化试剂的方案，其中许多试剂可在质量受控的商业试剂盒中获得。无论采用何种方案，要从生物组织中成功提取高质量核酸，都需要充分破坏组织和细胞结构、使核蛋白复合物变性、使核酸酶失活以及进行核酸纯化。这些步骤可根据目标核酸和生物样品来源进行修改。本章介绍了从多种样品和组织类型（包括唾液以及通常保存在临床病理实验室中的福尔马林固定石蜡包埋组织）中提取DNA和RNA的方法。还将讨论每种方案的特殊注意事项和常见陷阱，以及核酸定量技术。
