从兔股骨头中提取总RNA的方法特点

Method of extracting total RNA from rabbit femoral head

目的:建立一种高效、快速的骨组织总RNA提取方法。方法:实验于2005-01/2006-01在昆明医学院实验动物中心和中国科学院昆明动物研究所中科院细胞与分子进化重点实验室完成。取健康新西兰大白兔1只,截取其股骨头,迅速置于液氮罐中保存,于研钵中研磨,使骨组织始终保存于液氮中,继续研磨,如此重复3次,然后利用Trizol使骨细胞结构迅速破坏,将粉末转入离心管,室温静置5min。随后加入氯仿等有机溶剂处理、离心,使RNA与细胞DNA、蛋白质及其他成分分离从而得到总RNA。最后鉴定RNA的质量、纯度及产率,取2μL提取出的RNA在体积分数为0.008的甲醛变性琼脂糖凝胶上进行电泳,主要观察RNA的28S、18S及5S三个条带是否清晰,有无降解和DNA污染。以99μLDEPC水稀释1μLRNA样品,紫外分光光度计测量其吸光度(A)值,A260/A280之比值表示RNA的纯度,同时根据吸光度值计算其质量浓度。结果:①对提取的兔股骨头RNA进行琼脂糖凝胶电泳,可显示清晰的28S、18S两个条带,5S条带亦可见,表明了RNA是完整的。②用紫外分光光度法测定兔股骨头中提取出的RNAA260/A280,结果表明由本法提取的RNA纯度高,无DNA和蛋白质的污染。③经紫外分光光度计吸收定量,每毫克兔股骨头组织能提取1.0～1.2μg的总RNA。结论:本法提取骨组织总RNA方便、快捷,质量高,可用于骨组织的分子生物学研究。

AIM： To establish an efficient and quick method of acquiring total RNA from skeletal tissue. METHODS： The experiment was carried out from January 2005 to January 2006 in the ＂Experimental Animal Center of Kunming Medical College and the Key Laboratory of Cellular and Molecular Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences. One healthy New Zealand rabbit were adopted to intercept femoral head, which was immediately placed in liquid nitrogen tank for conservation and then grinded in morta for 3 times. After the cell construction was broken quickly by Trizol, the power was shifted into centrifuge tube for 5 minutes at room temperature, followed by additions of chloroform and other organic solvents as well as centrifugation. And then the total RNA was separated from DNA, protein and other tissues. The mass, purity and productive rate of RNA were evaluated. Degenerated sepharose electrophoresis was conducted on 2 μL extracted RNA by 0.008 volume fraction of formaldehyde to observe the three bands （28S, 18S and 5S） of RNA for their clarity, degradation .and DNA contamination. RNA sample of 1μL was diluted by 99μl. DEPC water, and the absorbance （A） was measured by ultraviolet spectrophotometer （UVS）. The ratio of A269/A280 was used to express the RNA purity, and the mass concentration was calculated according to A value. RESULTS： ①After the sepharose electrophoresis of RNA extracted from rabbit femoral head, the appearances of 5S band, clear 28S and clear 18S suggested that RNA was complete.②UVS result showed that the extracted RNA were in high purity without DNA and protein contamination. ③UVS result also showed that 1.0-1.2μg total RNA was acquired successfully from 1 mg skeletal tissue of rabbit femoral head. CONCLUSION： The method is a rapid and efficient purification for gaining total RNA from skeletal tissue, and it can be used for analyzing molecular biology of skeletal tissue.