改进TRIpure法提取小鼠肝脏组织总RNA

Isolation of Total RNA from Rat Liver Tissue by Improved Trizol Method

目的改进从新鲜动物组织中提取、鉴定纯度及完整性较高总RNA的技术方法。方法参考1987年ChomczynskiP等人提出的RNA提取方法,对组织RNA提取的操作步骤进行调整改进以获得稳定良好的RNA,并对常规琼脂糖凝胶电泳做适当改进用于鉴定RNA的质量。结果用该法提取的总RNA进行电泳后,可见28S、18S、5S三条清晰的RNA条带,其中28S条带的亮度约为18S条带的2倍,用紫外分光光度计检测RNA的纯度,OD260/OD280值为1.8～2.0,表明RNA没有被蛋白质或苯酚污染。这些结果显示RNA未降解并有较好的完整性,全部操作过程可在2h内完成。结论用改进方法提取的新鲜组织RNA质量稳定,电泳鉴定快速可靠。

Objective To improve a technique that is employed to isolate and identify high purity and integrity of total RNA extracted from fresh animal tissue. Methods Referring to the RNA isolation method proposed by Chomczynski P （1987）, we improved the protocol of RNA isolation. Furthermore, we adjusted the conventional agarose gel electrophoresis to be better used to identify the RNA quality. Results When total RNA, extracted by use of the method, was used for electrophoresis, it clearly showed three RNA bands of 28S, 18S, and 5S. The brightness of 28S RNA was twice as light as that of 18S RNA in the electrophoresis map. Purity analysis of total RNA was conducted with UV spectrophotometer. The value of OD260/OD280 varied from 1.8 to 2.0, which indicated that the RNA was not contaminated by proteins or phenol. The results indicated that the total RNA was complete and not degraded; and the entire procedure can be completed within 2 hrs. Conclusions The quality of RNA isolated from fresh tissue obtained by the improved method is stable, and electrophoresis method is rapid and reliable.