title: RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells.
abstract: Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. However, isolating bacterial RNA from infected cells or tissues is a challenging task, since mammalian RNA mostly dominates the lysates of infected cells. Here we describe an optimized method for RNA isolation of Listeria monocytogenes bacteria growing within bone marrow derived macrophage cells. Upon infection, cells are mildly lysed and rapidly filtered to discard most of the host proteins and RNA, while retaining intact bacteria. Next, bacterial RNA is isolated using hot phenol-SDS extraction followed by DNase treatment. The extracted RNA is suitable for gene transcription analysis by multiple techniques. This method is successfully employed in our studies of Listeria monocytogenes gene regulation during infection of macrophage cells (1-4). The protocol can be easily modified to study other bacterial pathogens and cell types.
authors.affiliation: The Department of Molecular Microbiology and Biotechnology, The George S. Wise Faculty of Life Sciences, Tel Aviv University.
authors.forename: Nadejda
authors.initials: N
authors.lastname: Sigal
authors.affiliation: The Department of Molecular Microbiology and Biotechnology, The George S. Wise Faculty of Life Sciences, Tel Aviv University.
authors.forename: Anna
authors.initials: A
authors.lastname: Pasechnek
authors.affiliation: The Department of Molecular Microbiology and Biotechnology, The George S. Wise Faculty of Life Sciences, Tel Aviv University; anathe@post.tau.ac.il.
authors.forename: Anat A
authors.initials: AA
authors.lastname: Herskovits
mesh_terms: D000818:Animals; D005786:Gene Expression Regulation; D008089:Listeria monocytogenes; D008264:Macrophages; D012329:RNA, Bacterial
chemical_list: D012329:RNA, Bacterial