Bacterial RNA isolation.
Bacterial RNA isolation.
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DOI：
10.1101/pdb.prot071068
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发表时间：
2012-09-01
影响因子：
--
通讯作者：
Ares, Manuel
中科院分区：
文献类型：
--
作者：
Ares, Manuel
在此细菌 RNA 分离方案中，“RNA 保护”处理之后是细胞壁肽聚糖成分的溶菌酶消化。 EDTA 促进革兰氏阴性菌外膜的损失，并使溶菌酶更好地接触肽聚糖。细胞在低渗溶菌酶缓冲液中消化期间开始裂解，并通过十二烷基硫酸钠 (SDS) 和热苯酚:氯仿:异戊醇 (PCA) 提取完成裂解。 SDS 和热苯酚会破坏细胞膜，使蛋白质（包括 RNase）变性，并从 RNA 中剥离蛋白质。使用锁相凝胶实现有机相与水相的分离，锁相凝胶是一种密度介于有机样品和水样品之间的惰性材料。样品分为三个相：从下到上，分别是苯酚和氯仿（有机相）、带有界面材料的惰性凝胶以及带有 RNA 的水相。凝胶充当样品和有机相及界面之间的物理屏障。有机提取后，通过乙醇沉淀浓缩 RNA。
In this bacterial RNA isolation protocol, an "RNA-protective" treatment is followed by lysozyme digestion of the peptidoglycan component of the cell wall. EDTA promotes the loss of the outer membrane of Gram-negative bacteria and allows the lysozyme better access to the peptidoglycan. Cells begin to lyse during digestion in hypotonic lysozyme buffer and lysis is completed by sodium dodecyl sulfate (SDS) and hot phenol:chloroform:isoamyl alcohol (PCA) extraction. SDS and hot phenol disrupt membranes, denature protein (including RNase), and strip proteins from RNA. The separation of the organic phase from the aqueous phase is achieved using Phase Lock Gel, an inert material with a density intermediate between the organic and aqueous samples. The sample is split into three phases: from bottom to top, these are phenol and chloroform (organic phase), the inert gel with the interface material, and the aqueous phase with the RNA. The gel acts as a physical barrier between the sample and the organic phase plus interface. Following organic extraction, the RNA is concentrated by ethanol precipitation.