Beltrame C O, Côrtes M F, Bandeira P T, Figueiredo A M S
Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil.
Braz J Med Biol Res. 2015 Dec;48(12):1071-6. doi: 10.1590/1414-431X20154734.
Biofilm formed by Staphylococcus aureus is considered an important virulence trait in the pathogenesis of infections associated with implantable medical devices. Gene expression analyses are important strategies for determining the mechanisms involved in production and regulation of biofilm. Obtaining intact RNA preparations is the first and most critical step for these studies. In this article, we describe an optimized protocol for obtaining total RNA from sessile cells of S. aureus using the RNeasy Mini Kit. This method essentially consists of a few steps, as follows: 1) addition of acetone-ethanol to sessile cells, 2) lysis with lysostaphin at 37°C/10 min, 3) vigorous mixing, 4) three cycles of freezing and thawing, and 5) purification of the lysate in the RNeasy column. This simple pre-kit procedure yields high-quality total RNA from planktonic and sessile cells of S. aureus.
金黄色葡萄球菌形成的生物膜被认为是与植入式医疗器械相关感染发病机制中的一个重要毒力特征。基因表达分析是确定生物膜产生和调控机制的重要策略。获得完整的RNA制剂是这些研究的首要也是最关键的步骤。在本文中，我们描述了一种使用RNeasy Mini试剂盒从金黄色葡萄球菌的固着细胞中获取总RNA的优化方案。该方法主要包括以下几个步骤：1）向固着细胞中加入丙酮 - 乙醇；2）在37°C下用溶葡萄球菌素裂解10分钟；3）剧烈混合；4）三个冻融循环；5）在RNeasy柱中纯化裂解物。这种简单的试剂盒前处理程序可从金黄色葡萄球菌的浮游细胞和固着细胞中获得高质量的总RNA。
