Raha S, Merante F, Proteau G, Reed J K
Department of Biochemistry, Erindale College, University of Toronto, Mississauga, Ontario, Canada.
Genet Anal Tech Appl. 1990 Nov;7(7):173-7. doi: 10.1016/0735-0651(90)90022-8.
A rapid procedure for the isolation of intact total cellular RNA from cultured cells is described. This method combines the simultaneous disruption of cells and extraction of nucleic acids in a single step with the use of phenol and a buffer containing 100 mM LiCl. Total cellular RNA can be isolated in approximately 2 hours. The yield and quality of the RNA is comparable to the more widely employed methods requiring extensive preparatory steps such as extraction using guanidinium thiocyanate and subsequent CsCl gradient centrifugation. The RNA isolated using our procedure contains transcripts up to 10 kb in length and is suitable for Northern analysis. This procedure also yields high-molecular-weight DNA, which is a suitable substrate for restriction endonucleases.
本文描述了一种从培养细胞中快速分离完整总细胞RNA的方法。该方法通过使用苯酚和含100 mM LiCl的缓冲液,在一步中同时实现细胞破碎和核酸提取。总细胞RNA可在约2小时内分离出来。RNA的产量和质量与需要大量制备步骤(如使用硫氰酸胍提取并随后进行CsCl梯度离心)的更广泛使用的方法相当。使用我们的方法分离的RNA包含长度达10 kb的转录本,适用于Northern分析。该方法还能产生高分子量DNA,这是限制性内切酶的合适底物。