Sharkey Freddie H, Banat Ibrahim M, Marchant Roger
School of Biological and Environmental Sciences, University of Ulster, BT52 1SA, Coleraine, Co. Londonderry, UK.
Extremophiles. 2004 Feb;8(1):73-7. doi: 10.1007/s00792-003-0363-2. Epub 2003 Nov 13.
Extraction of intact RNA is essential for quantitative gene expression analysis. Isolating high quality RNA from gram-positive bacteria is known to be problematic particularly from organisms that have optimal growth temperatures greater than 45 degrees C. We report a novel extraction protocol for the rapid isolation of fully intact RNA from thermophilic Geobacillus thermoleovorans using a lysing matrix containing a mixture of ceramic and glass beads, triisopropylnaphthalene sulfonic acid (TNS), and p-4-aminosalicyclic acid (PAS). Combining both detergents, TNS and PAS, appeared to increase denaturation of RNases at thermophilic temperatures. Gel electrophoresis revealed that only RNA isolated using the TNS-PAS procedure demonstrated sharp, undegraded 23S, 16S, and 5S ribosomal RNA bands. RNA extracted from geobacilli using commercially available kits was extensively degraded and was not suitable for detecting gene expression. Total RNA yields extracted with the TNS-PAS protocol were greater than eightfold higher than those obtained with available kits. Critically, it was also shown that only RNA isolated with the TNS-PAS-based method was suitable for monitoring thermophile gene expression patterns using RT-PCR analysis.
完整RNA的提取对于定量基因表达分析至关重要。已知从革兰氏阳性细菌中分离高质量RNA存在问题，特别是对于那些最佳生长温度高于45摄氏度的生物体。我们报告了一种新颖的提取方案，用于使用含有陶瓷和玻璃珠混合物、三异丙基萘磺酸（TNS）和对氨基水杨酸（PAS）的裂解基质从嗜热嗜热栖热放线菌中快速分离完全完整的RNA。将两种去污剂TNS和PAS结合使用，似乎可以增加嗜热温度下RNase的变性。凝胶电泳显示，只有使用TNS-PAS程序分离的RNA显示出清晰、未降解的23S、16S和5S核糖体RNA条带。使用市售试剂盒从地芽孢杆菌中提取的RNA被广泛降解，不适用于检测基因表达。用TNS-PAS方案提取的总RNA产量比使用现有试剂盒获得的产量高出八倍以上。至关重要的是，还表明只有用基于TNS-PAS的方法分离的RNA适用于使用RT-PCR分析监测嗜热菌基因表达模式。
