摘要
病原菌全基因组表达谱研究是阐明其致病机理的必要的基础 ,已经成为当前生命科学领域的热点和重点 ;然而由于难于从感染的组织中快速固定并分离细菌RNA ,从而极大的制约了该研究的进展。介绍一种从感染的细胞中分离细菌RNA的方法———冷酸酚法 ,其主要特点是 :(1)使用可以破碎真核细胞但不影响细菌细胞完整性的SDS浓度 ,实现了从感染的细胞中快速分离完整的细菌 ,减少了宿主细胞RNA的污染 ;(2 )在将细菌从细胞中分离的同时即利用苯酚 乙醇混合液将其总RNA快速固定 ,既减少了RNA的降解 ,又最大限度地保持了细菌在哺乳细胞内原有的表达模式 ;(3)可以从 10 8个菌体中提取到至少 30 μg的总RNA ,足够用于反转录等其他研究。该方法将为利用DNAMicroarray技术进行的病原菌表达谱研究提供有益的借鉴。
The cold-acidic phenol protocol—a new method that can isolate enough amounts of bacteria RNA from infected cells has been introduced in detail.At each time point, infected cells were lysed to recover the intracellular bacteria with 0.1% SDS, 1% acidic phenol and 19% ethanol in water for 30 min on ice. Bacteria were pooled and RNA was prepared using Promega SV total RNA purification kit according to manufacture's instruction. Bacterial RNAs were further purified by DNaseI treatment and phenol-chloroform extraction. Control RNAs from bacterial grown in L-broth and eukaryotic cells were isolated using the same RNA purification kit. RNA size chromatography was carried out with an Agilent 2100 Bioanalyser. This method is designed for quick lysis of host cells and preservation of bacterial RNA. The quality of RNA isolated from both cell-lines at all time points appeared to be excellent with minimal degradation and host RNA contamination. The quantity of the RNA from each time point of both cell-lines was enough for at least four times array hybridization.
出处
《微生物学报》
CAS
CSCD
北大核心
2004年第5期672-675,共4页
Acta Microbiologica Sinica
基金
国家重点基础研究发展规划项目 (G19990 5 410 3 )
国家"863计划"( 2 0 0 1AA2 2 3 0 11)
国家科技合作重点项目 ( 2 0 0 1AA2 2 3 116)~~
关键词
志贺氏菌
胞内细菌
RNA提取
冷酸酚法
Shigella spp., Intracellular bacteria, RNA isolation, Cold-acidic Phenol method
