Improved method for the isolation of RNA from bacteria refractory to disruption, including S. aureus producing biofilm
Improved method for the isolation of RNA from bacteria refractory to disruption, including S. aureus producing biofilm
复制标题
DOI：
10.1016/j.gene.2011.12.010
复制
发表时间：
2012-02-25
期刊：
影响因子：
3.5
通讯作者：
Ghaznavi-Rad, Ehsanollah
中科院分区：
文献类型：
--
作者：
Atshan, Salman Sahab;Shamsudin, Mariana Nor;Ghaznavi-Rad, Ehsanollah
介绍了一种快速、可靠、廉价的苯酚提取细菌生物膜产生菌RNA的方法。该方法在金黄色葡萄球菌和其他生物被膜革兰氏阴性微生物上进行了测试，与本文报道的其他方法相比，该方法提供了最高完整性的RNA回收。在平行实验中，使用标准方案使用Qigen、NucleoSpin RNAII、InnuREP RNA Mini、Trizol和MasterPure RNA提取试剂盒进行细菌裂解，始终得到低RNA产量，且缺乏完整性。用水煮法提取的RNA浓度较高，不含16S，含有235rRNA，含有5S RNA。由于改进了生物膜细菌细胞裂解，添加了热酚培养步骤，从而实现了更高的产量，而不需要珠磨机或酶。这种方法与Qiagen RNasy Mini试剂盒结合使用时，成功地提取了RNA，具有更高的完整性，包含未降解的16S和23S rRNA，不需要进一步纯化。DNA污染是RNA加工样品的一个问题；我们使用定量实时聚合酶链式反应(RT-qPCR)来测量从细菌生物膜细胞中回收RNA的方法。(C)2011爱思唯尔B.V.保留所有权利。
The development of fast, reliable and inexpensive phenol protocol is described for the isolation of RNA from bacterial biofilm producers. The method was tested on Staphylococcus aureus (S. aureus) and other biofilm-producing gram-negative microorganisms and provided the highest integrity of RNA recovery in comparison to other methods reported here. In parallel experiments, bacterial lysis with Qiagen, NucleoSpin RNAII, InnuREP RNA Mini, Trizol and MasterPure RNA extraction Kits using standard protocols consistently gave low RNA yields with an absence of integrity. The boiling method presented here yielded high concentration of RNA that was free from 16S and 235 rRNA, contained 5S RNA. Higher yields due to improved biofilm bacterial cell lysis were achieved with an added hot phenol incubation step without the need for a bead mill or the enzyme. This method when used in conjunction with the Qiagen RNeasy Mini kit, RNA isolation was a success with greater integrity and contained undegraded 16S and 23S rRNA and did not require further purification. Contaminating DNA was a problem with the RNA processing samples; we used quantitative real-time PCR (RT-qPCR) to measure the recovery of RNA from bacterial biofilm cells using the method described here. (C) 2011 Elsevier B.V. All rights reserved.