Abstract
Studying the transcriptome of bacterial pathogens during infection is a very informative and effective tool for discovering genes that contribute to successful infection. However, isolating bacterial RNA from infected cells or tissues is a challenging process due to the much higher amounts of host RNA in the lysates of infected cells. We have optimized a method for isolating RNA of Listeria monocytogenes (L. monocytogenes) bacteria infecting bone marrow derived macrophage cells (BMDM). After infection, we lyse the cells and filter the lysates through 0.45 µm filters to discard most of the host proteins and RNA. Next, we resuspend the bacteria and extract RNA following DNase treatment. The extracted RNA is suitable for gene expression analysis by real-time PCR or microarray. We have successfully employed this protocol in our studies of Listeria monocytogenes gene regulation during infection in vitro (Lobel et al., 2015; Lobel et al., 2012; Kaplan Zeevi et al., 2013; Rabinovich et al., 2012).
Keywords: Listeria monocytogenes, Bacterial RNA extraction, Intracellular bacterial RNA
Materials and Reagents
Equipment
Procedure
Representative data
We provide representative data of induction of the virulence gene hly of L. monocytogenes during intracellular growth in macrophages compared to growth in BHI medium (Figure 1). mRNA was purified from intracellular bacteria 6 hours post infection at M.O.I of 90:1 and from bacteria grown to mid log (OD600 = 0.5) in BHI medium. Figure 1. Transcription of hly during growth of L. monocytogenes in BMDMs (6 h.p.i.) and BHI lab medium. Error bars represent 95% confidence interval. Results are representative of 3 repeats (N=3). RQ: Relative quantity
Notes
Recipes
Acknowledgments
This protocol was adapted from the previously published studies (Lobel et al., 2015; Kaplan Zeevi et al., 2013; Lobel et al., 2012). This work was supported by the Israel Science Foundation and the ERA-NET PathoGenoMics (funded by MOH Israel) grants to AAH. L.L is supported by the Argentinian friends of TAU.
References
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