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Isolation of Brucella abortus total RNA from B. abortus-infected murine RAW macrophages.

Abstract：

:Brucella is a Gram-negative facultative bacterium that persists intracellularly in macrophages. However, the intracellular survival mechanisms used by Brucella are not fully understood. Isolation of Brucella RNA from infected macrophages has been challenging, and the inability to isolate sufficient Brucella RNA from infected macrophages has contributed to the failure in understanding bacterial transcriptional events. We describe the isolation of sufficient Brucella abortus RNA from its infective host cell environment using osmotic lysis and RNase and DNase digestion. This method takes advantage of the B. abortus cell envelope that protects bacterial RNA and DNA. The cell envelope of B. abortus was digested using SDS/proteinase K (PK) that, importantly, inhibits any residual RNase after digesting macrophage RNA permitting the extraction of B. abortus RNA. In our experiments, 4.5 microg of RNA was routinely isolated from 1 ml bacterial culture and 2-9 microg of bacterial RNA from infected macrophages without detectable host cell RNA or DNA contamination. The method is rapid and uses inexpensive, commonly available reagents. Total bacterial RNA was isolated in quantities sufficient for RT-PCR and microarray analysis.

journal_name

J Microbiol Methods

journal_title

Journal of microbiological methods

authors

Covert J,Eskra L,Splitter G

doi

10.1016/j.mimet.2004.10.018

subject

Has Abstract

pub_date

2005-03-01 00:00:00

pages

383-93

issue

3

eissn

0167-7012

issn

1872-8359

pii

S0167-7012(04)00301-X

journal_volume

60

pub_type

杂志文章

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