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

, : Isolation of Brucella abortus total RNA from B. abortus-infected murine RAW macrophages. Journal of Microbiological Methods 60(3): 383-393

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 1 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 micro g of RNA was routinely isolated from 1 ml bacterial culture and 2-9 micro g 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.


PMID: 15649540

DOI: 10.1016/j.mimet.2004.10.018

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Loo, E.; Magis, R.C.; Santarriaga, S.M., 1996: Sentinel serosurveys in Mexico 1990-1994. ELEVENTH INTERNATIONAL CONFERENCE ON AIDS Eleventh International Conference on AIDS, Vol Two One world: One hope : 463

Anjum, S.; Doucet, A.; Holmes, C.C., 2010: A boosting approach to structure learning of graphs with and without prior knowledge. Identifying the network structure through which genes and their products interact can help to elucidate normal cell physiology as well as the genetic architecture of pathological phenotypes. Recently, a number of gene network inference tools have...

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Thomson, C.G.; Sircus, W., 1968: Effects of SC 15396 on gastric secretion. 3. The action in the rat. Gut 9(6): 649-654

Smith, S.R., 1987: Tolerance--a response to long-term treatment with beta 2-receptor stimulants. Journal of Clinical Pharmacy and Therapeutics 12(3): 137-139

Barber, M.; Nellen, M., 2010: Erysipeloid of Rosenbach; response to penicillin. Lancet 1(6387): 125-127

W.M.L.; Hanlin R.T., 1992: Ascomal development in leptosphaerulina crassiasca. Cultures of Leptosphaerulina crassiasca from single ascospores formed ascostromata, indicating that the fungus is homothallic. Mycelial hyphae were septate, with uninucleate cells, and they gave rise to fertile hyphae which were larger and had bin...

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