Cheley S, Anderson R
Anal Biochem. 1984 Feb;137(1):15-9. doi: 10.1016/0003-2697(84)90339-7.
A rapid, microanalytical procedure for the reproducible isolation of RNA from small cultured cell samples and application to dot-blot hybridization is described. The procedure employs guanidine hydrochloride solubilization of whole cells, disruption by syringing, and selective precipitation of RNA with ethanol. The method can be performed in a single tissue culture tube and obviates the need for removal of nuclei or for organic solvent extractions. Recovery of RNA from small cell samples (10(6) cells) is 51%, while 97% of the DNA and 99% of the protein are eliminated by the procedure. Detection of specific RNA by dot-blot hybridization using a labeled probe demonstrates high reproducibility of recovered RNA and lack of "masking" with up to a 10-fold excess of starting cell material. Applicability of the procedure to detection of virus-specific RNA in cells persistently infected with mouse hepatitis virus is described.
本文描述了一种从少量培养细胞样本中可重复分离RNA并应用于点杂交的快速微量分析方法。该方法采用盐酸胍溶解全细胞，通过注射器破碎细胞，并使用乙醇选择性沉淀RNA。该方法可在单个组织培养管中进行，无需去除细胞核或进行有机溶剂提取。从小细胞样本（10⁶个细胞）中回收RNA的效率为51%，而该方法可去除97%的DNA和99%的蛋白质。使用标记探针通过点杂交检测特定RNA，结果表明回收的RNA具有高度可重复性，并且即使起始细胞材料过量10倍也不会出现“掩盖”现象。本文还描述了该方法在检测持续感染小鼠肝炎病毒的细胞中病毒特异性RNA的适用性。
