Nuyts S, Van Mellaert L, Lambin P, Anné J
Laboratory of Bacteriology, Rega Institute, K.U. Leuven, Minderbroedersstraat 10, 3000 Leuven, Belgium.
J Microbiol Methods. 2001 Apr;44(3):235-8. doi: 10.1016/s0167-7012(01)00219-6.
Several molecular techniques require high-quality RNA, free from DNA. Various methods have been described to obtain RNA to be used in expression studies or as starting material in differential display-reverse transcriptase (dd-RT-PCR), for which high-quality RNA free from DNA is an essential requirement. In this report, we compare three different methods to isolate RNA from Gram-positive bacteria: (1) An acid-phenol extraction protocol. (2) The "RNeasy mini kit" from QIAGEN (Valencia, CA, USA). (3) The "SV Total RNA Isolation System" from Promega (Madison, WI, USA).The QIAGEN-kit delivers the highest amount of RNA with the highest purity. Slot blot analysis and dd-RT-PCR confirm the absence of DNA contamination and Northern blot analysis and dd-RT-PCR show high quality of the extracted RNA. This RNA extraction method thus addresses current problems by permitting rapid and safe isolation with high yields of intact RNA for subsequent analysis.
几种分子技术需要高质量的、无DNA污染的RNA。为了获得用于表达研究或作为差异显示逆转录酶（dd-RT-PCR）起始材料的RNA，人们已经描述了各种方法，对于这些方法而言，无DNA污染的高质量RNA是一项基本要求。在本报告中，我们比较了从革兰氏阳性菌中分离RNA的三种不同方法：（1）酸酚提取方案。（2）美国加利福尼亚州瓦伦西亚的QIAGEN公司生产的“RNeasy迷你试剂盒”。（3）美国威斯康星州麦迪逊的Promega公司生产的“SV总RNA分离系统”。QIAGEN试剂盒提取的RNA量最高，纯度也最高。狭缝印迹分析和dd-RT-PCR证实不存在DNA污染，Northern印迹分析和dd-RT-PCR表明提取的RNA质量很高。因此，这种RNA提取方法通过允许快速、安全地分离出高产量的完整RNA用于后续分析，解决了当前的问题。
