title: 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.
authors.affiliation: Laboratory of Molecular and Cellular Immunology, School of Veterinary Medicine, Department of Animal Health and Biomedical Sciences, 1656 Linden Dr., University of Wisconsin-Madison, Madison, WI 53706, USA.
authors.forename: Jill
authors.initials: J
authors.lastname: Covert
authors.forename: Linda
authors.initials: L
authors.lastname: Eskra
authors.forename: Gary
authors.initials: G
authors.lastname: Splitter
mesh_terms: D000818:Animals; D002003:Brucella abortus / Q000235:genetics* / Q000254:growth & development; D002006:Brucellosis / Q000382:microbiology*; D002460:Cell Line; D015169:Colony Count, Microbial; D003851:Deoxyribonucleases / Q000378:metabolism; D008264:Macrophages / Q000382:microbiology*; D051379:Mice; D009995:Osmosis; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012260:Ribonucleases / Q000378:metabolism
chemical_list: D012329:RNA, Bacterial; D003851:Deoxyribonucleases; D012260:Ribonucleases