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Immunology and Infection

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Published: June 4th, 2016



1The Department of Molecular Microbiology and Biotechnology, The George S. Wise Faculty of Life Sciences, Tel Aviv University

Here we describe a method for bacterial RNA isolation from Listeria monocytogenes bacteria growing inside murine macrophages. This technique can be used with other intracellular pathogens and mammalian host cells.

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.

Intracellular bacterial pathogens ─ bacteria causing infectious diseases and capable of growing and reproducing inside cells of human hosts ─ are a major health concern worldwide5. To invade and replicate within a mammalian cell, intracellular pathogens have acquired sophisticated virulence mechanisms and factors. While these mechanisms are fundamental to the ability to cause a disease, we know little about their regulation and dynamics. Since gene expression profiles of bacteria grown in liquid media do not reflect the actual environment within host cells, there is a growing need for transcriptome analyses of bacteria grown in their intracellul....

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Note: During the entire experiment, macrophage cells are incubated at 37 °C in a 5% CO2 forced-air incubator and taken out of the incubator only for experimental manipulations, which are performed in a Class II biological safety cabinet. Working with L. monocytogenes bacteria is according to biological safety level 2 regulations.

1. Cell Preparation and Bacterial Infection (Day 1 and 2)

  1. Day 1
    1. Seed 2.0 x 107 bone marrow derived macrophage cells (BMDM) on a.......

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The model system is shown in Figure 1 and includes macrophage cells infected with L. monocytogenes bacteria, which replicate in the macrophage cytosol. Figure 2 represents the experimental scheme. Figure 3 represents typical results of such RT-qPCR analysis of virulence genes during WT L. monocytogenes growth in macrophages in comparison to growth in rich laboratory medium BHI. The results show the transcription levels o.......

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The protocol described here represents an optimized method for isolation of bacterial RNA from L. monocytogenes bacteria growing intracellularly in macrophage cells. This protocol is based on cell differential lysis and includes two major steps for enrichment of bacterial RNA: macrophage nuclei sedimentation using centrifugation and a rapid collection of bacteria by filtration. These steps are followed by a standard RNA extraction procedure. While this protocol describes purification of listerial RNA, it can be .......

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The research in the Herskovits lab is supported by 335400 ERC and R01A/109048 NIH grants.


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Name Company Catalog Number Comments
Listeria monocytogenes 10403S 20
Bone marrow derived macrophages prepared from C57B/6 female mice 21
H2O, RNAse free Thermo Scientific 10977-015 DEPC-treated water can be used
DMEM Gibco 41965039
Glutamine Gibco 25030081
Sodium pyruvate Gibco 11360-088
b-Mercaptoethanol   Gibco 31350010
Pen/Strep Gibco 15140-122
Gentamicin Sigma-Aldrich G1397
FBS Gibco 10270106
Dulbecco’s Phosphate Buffered Saline-PBS Sigma-Aldrich D8537
Brain heart infusion (BHI) Merckmillipore 1104930500
Phenol saturated pH 4.3 Fisher BP1751I-400
Chloroform Fisher BP1145-1
Iso-amyl alcohol Sigma-Aldrich W205702
Sodium acetate Sigma-Aldrich W302406
EDTA Sigma-Aldrich EDS
DNaseI Fermentas, EN0521
SDS 10% Sigma-Aldrich L4522
Ethanol absolute Merck Millipore 1070174000
37°C, 5% CO2 forced-air incubator Thermo Scientific Model 3111
Cell scrapers Nunc 179693
Kontes glass holder for 45 mm filters Fisher K953755-0045
MF-Millipore filters 45 mm, 0.45 µm Merck Millipore HAWP04700
SpeedVac system Thermo Scientific SPD131DDA
Vortex-Genie 2 Scientific Industries Model G560E
NanoDrop Thermo Scientific
145 mm cell culture dishes Greiner 639 160
1.7 ml tubes, RNase-free Axygen MCT-175-C
30°C incubator Thermo Scientific
65 °C heat block Thermo Scientific
4 °C table centrifuge Eppendorf 5417R
Sterile pipettes, 25 ml Greiner
Falcon tubes, 50 ml Greiner
Liquid nitrogen

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