The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something years on
The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something years on
复制标题
DOI：
10.1038/nprot.2006.83
复制
发表时间：
2006-01-01
期刊：
影响因子：
14.8
通讯作者：
Sacchi, Nicoletta
中科院分区：
文献类型：
--
作者：
Chomczynski, Piotr;Sacchi, Nicoletta
自引入以来，“一步法”已被广泛用于从不同来源的生物样品中分离总RNA。该方法的基本原理是，在用含有硫氰酸胍、乙酸钠、苯酚和氯仿的酸性溶液提取后，将RNA与DNA分离，然后离心。在酸性条件下，总RNA保留在上层水相中，而大部分DNA和蛋白质保留在界面或下层有机相中。然后通过用异丙醇沉淀回收总RNA，并可用于多种应用。最初的方案，能够在不到4小时的时间内从细胞和组织中分离RNA，极大地推进了植物和动物模型以及病理样品中基因表达的分析，正如该论文在20多年来获得的大量引用所证明的那样。
Since its introduction, the 'single-step' method has become widely used for isolating total RNA from biological samples of different sources. The principle at the basis of the method is that RNA is separated from DNA after extraction with an acidic solution containing guanidinium thiocyanate, sodium acetate, phenol and chloroform, followed by centrifugation. Under acidic conditions, total RNA remains in the upper aqueous phase, while most of DNA and proteins remain either in the interphase or in the lower organic phase. Total RNA is then recovered by precipitation with isopropanol and can be used for several applications. The original protocol, enabling the isolation of RNA from cells and tissues in less than 4 hours, greatly advanced the analysis of gene expression in plant and animal models as well as in pathological samples, as demonstrated by the overwhelming number of citations the paper gained over 20 years.