Name: RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells (Video) | JoVE Journal
Uploaded: 2016-06-04T14:00:00.000+00:00
Duration: 10 min 42 s
Description: Learn RNA purification from intracellular Listeria monocytogenes in macrophage cells. This method enriches bacterial RNA while minimizing host contamination for transcriptomic analysis.

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5 minutes

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

Note: During the entire experiment, macrophage cells are incubated at 37 &#176;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)
<ol>
	<li>Day 1
	<ol>
		<li>Seed 2.0 x 107 bone marrow derived macrophage cells (BMDM) on a 145 mm dish in 30 ml BMDM + Pen-Strep media (Table 2). Seed 3 plates for each bacterial strain to be analyzed. Incubate overnight at 37 &#176;C in a 5% CO2 forced-air incubator.</li>
		<li>Start an overnight bacterial culture of wild-type (WT) L. monocytogenes strain 10403S in 10 ml of brain heart infusion (BHI) medium, at 30 &#176;C in a standard incubator. Place culture tubes slanted without shaking. Note: These growth conditions up-regulate the expression of the flagella genes, which promotes efficient infection.</li>
	</ol>
	</li>
	<li>Day 2
	<ol>
		<li>Pre-warm BMDM medium (without Pen-Strep antibiotics) (Table 2) and phosphate buffered saline (PBS) at 37 &#176;C.</li>
		<li>Wash macrophage monolayer twice with 25 ml of pre-warmed PBS to remove antibiotics. Add 30 ml fresh BMDM medium.</li>
		<li>Wash 1.5 ml of overnight bacterial culture with PBS by centrifuging bacteria at&#160; &#62; 14,000 x g for 1 min, discarding the supernatant, and resuspending bacteria gently in 1.5 ml of PBS by pipetting. Repeat twice.</li>
		<li>Infect each macrophage plate with 0.5 ml of a washed overnight culture of wild-type L. monocytogenes. If using multiple plates, infect each plate 15 min apart. This time interval will enable harvesting each plate individually at the end of the infection. 
		Note: The multiplicity of infection (MOI) is assayed by plating serial dilutions of bacterial culture used for infection on BHI agar plates and counting colony forming units (CFU) after 24 hr incubation of plates at 37 &#176;C. Using 0.5 ml of WT L. monocytogenes strain 10403S results in a MOI of ~ 100 (100 bacteria CFU per macrophage cell). When analyzing bacterial mutants defective in intracellular growth, a higher MOI should be considered.</li>
		<li>Following 0.5 hr incubation at 37 &#176;C, wash the infected cells twice with PBS, to remove unattached bacteria, and add 30 ml pre-warmed BMDM medium. Attached bacteria will internalize during next 0.5 hr.</li>
		<li>At 1 hr post infection, add 30 &#181;l of gentamicin (1:1,000 to reach a final concentration of 50 &#181;g/ml) to kill extra-cellular bacteria.</li>
		<li>Assemble the filter apparatus by placing a filter head on a collecting liquid flask with a vacuum outlet port. Then place a filter (0.45 &#181;m) on a filter head, followed by a cylinder funnel and secure the different parts with a metal clamp. Prepare ice-cold RNase-free water.</li>
		<li>At 6 hr post-infection, harvest bacteria from infected cells as follows. Treat each plate individually. 
		Note: Usually, L. monocytogenes completes 1-log of growth during 6 hr of infection in macrophage cells. Longer incubations could result in bacterial overgrowth and cell death of the macrophages, while shorter incubation periods could result in considerably lower bacterial loads. The MOI and the time of infection can be modified to reach optimal conditions.
		<ol>
			<li>Wash infected cells with PBS once. Add 20 ml of ice-cold RNase-free water to lyse macrophage cells. Using a cell scraper, scrape the cells off the plate quickly but carefully.</li>
			<li>Collect lysed cells to a 50 ml conical tube. Vortex for 30 sec. Centrifuge at 800 x g for 3 min at 4 &#176;C.</li>
			<li>Pass the supernatant through the filter apparatus using a vacuum system. Using tweezers, roll the filter and quickly transfer it to a 15 ml conical tube. Snap-freeze the tubes with the filters in liquid nitrogen.</li>
			<li>Assemble the filtration apparatus with a new filter for the next sample, as described in 1.2.7.</li>
			<li>Store the frozen filters at -80 &#176;C for the next day. Alternatively, proceed directly to nucleic acid extraction.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Nucleic Acids Extraction (Day 3)
Note: Perform all manipulations with phenol and chloroform solutions in a fume hood.
<ol>
	<li>Prepare a 1:1 mix of acidified phenol: chloroform, 400 &#181;l for each sample. Mix in separate tubes, and then withdraw the resulting aqueous layer by aspiration. Add 40 &#181;l of 10% SDS.</li>
	<li>Thaw the filter-containing tubes on ice to keep them cold. To each filter-containing tube add 650 &#181;l of acetate-EDTA (AE) buffer (Table 2).</li>
	<li>Perform the following steps as quickly as possible.
	<ol>
		<li>Vigorously vortex the tube containing the filter so that the filter whisks to the periphery of the tube and the buffer fully washes the filter. Always keep the tube cold by placing it back on ice. Note: It may be necessary to additionally vortex the tube while inverted to fully wash the bacteria off the filter.</li>
		<li>Repeat for all tubes. It may be useful to spin-down the suspension shortly (1 min at 120 x g) at the end of the process.</li>
	</ol>
	</li>
	<li>Transfer the bacteria-containing buffer to a 1.5 ml microcentrifuge tube containing the SDS and phenol/chloroform mix (as prepared in 2.1). Repeat for all tubes. It may be necessary to spin the filter containing tubes (1 min at 120 x g) again to get residual buffer from the filter.</li>
	<li>Place all the 1.5 ml tubes in a multi-tube vortex device, and vortex at full speed for 10 min.</li>
	<li>Incubate the tubes in a 65 &#176;C heat block for 10 min. Centrifuge at maximum speed (&#62; 14,000 x g) for 5 min.</li>
	<li>Transfer the aqueous layer (about 400 &#181;l) from each tube to a new 1.5 ml tube containing 40 &#181;l of 3 M sodium acetate (pH 5.2) and 1.0 ml of 100% ethanol. Vortex each tube thoroughly. 
	Note: Glycogen can be added at this step to improve visualization of the precipitated pellet.</li>
	<li>Incubate the samples at -80 &#176;C for 1 hr. Alternatively, incubate samples at -20 &#176;C overnight. Then, centrifuge the samples at 4 &#176;C for 20 min at maximum speed (&#62; 14,000 x g).</li>
	<li>Carefully aspirate off the ethanol (upper layer) from each tube. Add 500 &#181;l cold 70% ethanol to each sample and vortex thoroughly. Centrifuge at 4 &#176;C for 20 min at maximum speed (&#62; 1 4,000 x g).</li>
	<li>Carefully aspirate off the ethanol from each tube. Dry the samples for approximately 2 min using a vacuum evaporator with no heat. Do not over-dry the samples.</li>
	<li>Add 25 &#181;l RNase-free water to each sample. Incubate at room temperature for 20 min. Carefully vortex and spin-down.</li>
	<li>Measure RNA concentration in the samples using a microvolume UV-Vis spectrophotometer. Expect to extract about 0.5 - 1 &#181;g of nucleic acids per plate. 
	Note: Technical replicates can be combined at this stage.</li>
</ol>
3. DNase Treatment
<ol>
	<li>Set up the reaction according to Table 1 in a microfuge tube. Incubate at 37 &#176;C for 45 min. Add 450 &#181;l RNase-free water and 500 &#181;l of Phenol-Chloroform-IAA mix (Table 2), separate phases by centrifuging for 2 min at maximal speed (&#62; 14,000 x g).</li>
	<li>Transfer the aqueous layer to a new tube and add 500 &#181;l chloroform-IAA mix (Table 2), vortex and separate phases by 2 min centrifugation at maximal speed (&#62; 14,000 x g).</li>
	<li>Transfer the aqueous layer to a new tube and add 1 ml of ethanol and 50 &#181;l of 3 M sodium acetate (pH 5.2). Vortex. 
	Note: Glycogen can be added at this step to improve visualization of the precipitated pellet.</li>
	<li>Incubate for 1 hr at -80 &#176;C. Alternatively, incubate samples at -20 &#176;C overnight. Centrifuge at 4 &#176;C for 20 min at maximal speed.</li>
	<li>Carefully aspirate off the ethanol from each tube. Add 500 &#181;l cold 70% ethanol to each sample and vortex thoroughly. Centrifuge at 4 &#176;C for 20 min at maximum speed. Carefully aspirate off the ethanol from each tube.</li>
	<li>Dry the samples for approximately 2 min using a vacuum evaporator with no heat. Do not over-dry the samples.</li>
	<li>Add 12 &#181;l of RNase-free water, incubate for 2 min at room temperature, vortex, and spin-down. The samples contain purified RNA, keep them on ice. Alternatively, dissolve RNA in RNase-free H2O with 0.1 mM EDTA or TE buffer (10 mM Tris, 1 mM EDTA).</li>
	<li>Measure RNA concentrations using a microvolume UV-Vis spectrophotometer. Expect ~ 100 ng of RNA per sample (if technical replicates are combined). 
	Note: RNA extracted according to this protocol is suitable for gene transcription analysis by multiple techniques. We usually employ RT-qPCR analysis to study L. monocytogenes gene transcription during infection of macrophage cells 1-4.</li>
</ol>

<ol><li>Lobel, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+metabolic+regulator+CodY+links+Listeria+monocytogenes+metabolism+to+virulence+by+directly+activating+the+virulence+regulatory+gene+prfA%5BTitle%5D)+AND+%22Mol+Microbiol%22%5BJournal%5D)">The metabolic regulator CodY links Listeria monocytogenes metabolism to virulence by directly activating the virulence regulatory gene prfA.</a> Mol Microbiol. , (2014).</li>
<li>Lobel, L., Sigal, N., Borovok, I., Ruppin, E., Herskovits, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Integrative+genomic+analysis+identifies+isoleucine+and+CodY+as+regulators+of+Listeria+monocytogenes+virulence%5BTitle%5D)+AND+%22PLoS+Genet%22%5BJournal%5D)">Integrative genomic analysis identifies isoleucine and CodY as regulators of Listeria monocytogenes virulence.</a> PLoS Genet. 8 , e1002887(2012).</li>
<li>Kaplan Zeevi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Listeria+monocytogenes+multidrug+resistance+transporters+and+cyclic+di-AMP%2C+which+contribute+to+type+I+interferon+induction%2C+play+a+role+in+cell+wall+stress%5BTitle%5D)+AND+%22J+Bacteriol%22%5BJournal%5D)">Listeria monocytogenes multidrug resistance transporters and cyclic di-AMP, which contribute to type I interferon induction, play a role in cell wall stress.</a> J Bacteriol. 195, 5250-5261 (2013).</li>
<li>Rabinovich, L., Sigal, N., Borovok, I., Nir-Paz, R., Herskovits, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prophage+excision+activates+Listeria+competence+genes+that+promote+phagosomal+escape+and+virulence%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Prophage excision activates Listeria competence genes that promote phagosomal escape and virulence.</a> Cell. 150, 792-802 (2012).</li>
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<li>Swaminathan, B., Gerner-Smidt, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+epidemiology+of+human+listeriosis%5BTitle%5D)+AND+%22Microbes+Infect%22%5BJournal%5D)">The epidemiology of human listeriosis.</a> Microbes Infect. 9, 1236-1243 (2007).</li>
<li>Hamon, M., Bierne, H., Cossart, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Listeria+monocytogenes%3A+a+multifaceted+model%5BTitle%5D)+AND+%22Nat+Rev+Microbiol%22%5BJournal%5D)">Listeria monocytogenes: a multifaceted model.</a> Nat Rev Microbiol. 4, 423-434 (2006).</li>
<li>Dussurget, O., Pizarro-Cerda, J., Cossart, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+determinants+of+listeria+monocytogenes+virulence%5BTitle%5D)+AND+%22Ann+Rev+Microbiol%22%5BJournal%5D)">Molecular determinants of listeria monocytogenes virulence.</a> Ann Rev Microbiol. 58, 587-610 (2004).</li>
<li>Portnoy, D. A., Auerbuch, V., Glomski, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+cell+biology+of+Listeria+monocytogenes+infection%3A+the+intersection+of+bacterial+pathogenesis+and+cell-mediated+immunity%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">The cell biology of Listeria monocytogenes infection: the intersection of bacterial pathogenesis and cell-mediated immunity.</a> J Cell Biol. 158, 409-414 (2002).</li>
<li>Freitag, N. E., Port, G. C., Miner, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Listeria+monocytogenes+-+from+saprophyte+to+intracellular+pathogen%5BTitle%5D)+AND+%22Nat+Rev+Microbiol%22%5BJournal%5D)">Listeria monocytogenes - from saprophyte to intracellular pathogen.</a> Nat Rev Microbiol. 7, 623-628 (2009).</li>
<li>Graham, J. E., Clark-Curtiss, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+Mycobacterium+tuberculosis+RNAs+synthesized+in+response+to+phagocytosis+by+human+macrophages+by+selective+capture+of+transcribed+sequences+%28SCOTS%29%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Identification of Mycobacterium tuberculosis RNAs synthesized in response to phagocytosis by human macrophages by selective capture of transcribed sequences (SCOTS).</a> Proc Natl Acad Sci U S A. 96, 11554-11559 (1999).</li>
<li>Toledo-Arana, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Listeria+transcriptional+landscape+from+saprophytism+to+virulence%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">The Listeria transcriptional landscape from saprophytism to virulence.</a> Nature. 459, 950-956 (2009).</li>
<li>Schnappinger, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transcriptional+Adaptation+of+Mycobacterium+tuberculosis+within+Macrophages%3A+Insights+into+the+Phagosomal+Environment%5BTitle%5D)+AND+%22J+Exp+Med%22%5BJournal%5D)">Transcriptional Adaptation of Mycobacterium tuberculosis within Macrophages: Insights into the Phagosomal Environment.</a> J Exp Med. 198, 693-704 (2003).</li>
<li>Albrecht, M., Sharma, C. M., Reinhardt, R., Vogel, J., Rudel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Deep+sequencing-based+discovery+of+the+Chlamydia+trachomatis+transcriptome%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">Deep sequencing-based discovery of the Chlamydia trachomatis transcriptome.</a> Nucleic Acids Res. 38, 868-877 (2010).</li>
<li>La, M. V., Raoult, D., Renesto, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulation+of+whole+bacterial+pathogen+transcription+within+infected+hosts%5BTitle%5D)+AND+%22FEMS+Microbiol+Rev%22%5BJournal%5D)">Regulation of whole bacterial pathogen transcription within infected hosts.</a> FEMS Microbiol Rev. 32, 440-460 (2008).</li>
<li>Westermann, A. J., Gorski, S. A., Vogel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dual+RNA-seq+of+pathogen+and+host%5BTitle%5D)+AND+%22Nat+Rev+Microbiol%22%5BJournal%5D)">Dual RNA-seq of pathogen and host.</a> Nat Rev Microbiol. 10, 618-630 (2012).</li>
<li>Rienksma, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comprehensive+insights+into+transcriptional+adaptation+of+intracellular+mycobacteria+by+microbe-enriched+dual+RNA+sequencing%5BTitle%5D)+AND+%22BMC+Genomics%22%5BJournal%5D)">Comprehensive insights into transcriptional adaptation of intracellular mycobacteria by microbe-enriched dual RNA sequencing.</a> BMC Genomics. 16, 34(2015).</li>
<li>Afonso-Grunz, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dual+3%27Seq+using+deepSuperSAGE+uncovers+transcriptomes+of+interacting+Salmonella+enterica+Typhimurium+and+human+host+cells%5BTitle%5D)+AND+%22BMC+Genomics%22%5BJournal%5D)">Dual 3'Seq using deepSuperSAGE uncovers transcriptomes of interacting Salmonella enterica Typhimurium and human host cells.</a> BMC Genomics. 16, 323(2015).</li>
<li>Englen, M. D., Valdez, Y. E., Lehnert, N. M., Lehnert, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Granulocyte%2Fmacrophage+colony-stimulating+factor+is+expressed+and+secreted+in+cultures+of+murine+L929+cells%5BTitle%5D)+AND+%22J+Immunol+Methods%22%5BJournal%5D)">Granulocyte/macrophage colony-stimulating factor is expressed and secreted in cultures of murine L929 cells.</a> J Immunol Methods. 184, 281-283 (1995).</li>
<li>Becavin, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+widely+used+Listeria+monocytogenes+strains+EGD%2C+10403S%2C+and+EGD-e+highlights+genomic+variations+underlying+differences+in+pathogenicity%5BTitle%5D)+AND+%22MBio%22%5BJournal%5D)">Comparison of widely used Listeria monocytogenes strains EGD, 10403S, and EGD-e highlights genomic variations underlying differences in pathogenicity.</a> MBio. 5, e00969-e900914 (2014).</li>
<li>Celada, A., Gray, P. W., Rinderknecht, E., Schreiber, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evidence+for+a+gamma-interferon+receptor+that+regulates+macrophage+tumoricidal+activity%5BTitle%5D)+AND+%22J+Exp+Med%22%5BJournal%5D)">Evidence for a gamma-interferon receptor that regulates macrophage tumoricidal activity.</a> J Exp Med. 160, 55-74 (1984).</li>
</ol>

The authors have nothing to disclose.

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 ...

Intracellular bacterial pathogens &#9472; bacteria causing infectious diseases and capable of growing and reproducing inside cells of human hosts &#9472; 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 intracellular niches. Such analyses will enable the deciphering of specific bacterial adaptations triggered by the host and will help to identify new targets for therapeutic design. Transcriptome analysis of intracellularly grown bacteria is highly challenging since mammalian RNA outnumber bacterial RNA by at least ten fold. In this manuscript, we describe an experimental method to isolate bacterial RNA from Listeria monocytogenes bacteria growing inside murine macrophage cells. The extracted RNA can be used to study intracellular adaptations and virulence mechanisms of pathogenic bacteria by various techniques of transcription analysis, such as RT-PCR, RNA-Seq, microarray and other hybridization based technologies.
L. monocytogenes is the causative agent of listeriosis in humans, a disease with clinical manifestations targeting primarily immunocompromised individuals, elderly people and pregnant women6. It is a Gram-positive facultative intracellular pathogen that invades a wide array of mammalian cells that have been used for decades as a model in host-pathogen interactions studies7. Upon invasion, it resides initially in a vacuole or a phagosome (in the case of phagocytic cells), from which it must escape into the host cell cytosol in order to replicate. Several virulence factors have been shown to mediate the escape process, primarily the pore-forming hemolysin, Listeriolysin O (LLO) and two additional phospholipases8. In the cytosol the bacteria use the host actin polymerization machinery to propel themselves on actin filaments within the cell and to spread from cell to cell (Figure 1). All major virulence factors of L. monocytogenes involved in invasion, intracellular survival and replication, are activated by the master virulence transcription regulator, PrfA. 8-10.
In the last decade, several studies conducted by us and others have successfully applied methods for transcriptome analysis of intracellularly grown bacteria inside host cells2,11-15. Two main approaches are used to separate bacterial RNA from host-RNA which are based on: 1) Selective enrichment of bacterial RNA and 2) RNA isolation by differential cell lysis. The first approach relies on subtractive hybridization of total RNA extracts to mammalian RNA molecules (for example using commercially available kits) or selective capture of bacterial transcribed sequences (SCOTS)11. The second approach relies on differential lysis of bacteria and host cells, in which the host cells are lysed while bacterial cells remain intact. Bacterial cells are then separated from the host cell lysate, usually by centrifugation, and the RNA is extracted using standard techniques. The main problem using this approach is that together with the intact bacteria, host cells nuclei are also isolated, thus the RNA preparations still contain mammalian RNA. One way to overcome this problem is to separate intact bacteria from host cells nuclei using differential centrifugation, though this procedure usually takes time raising the concern of changes in gene expression profile during extraction. In this paper we present an improved and rapid bacteria RNA extraction protocol, which is based on cell differential lysis approach. First, L. monocytogenes infected macrophage cells are lysed with cold water. Next, macrophage nuclei are removed by a brief centrifugation and intact bacteria are rapidly collected on filters, from which RNA is isolated using hot phenol-SDS extraction of bacterial nucleic acids.

<table><tbody><tr><td>Listeria monocytogenes 10403S</td><td></td><td></td><td>&lt;sup&gt;20&lt;/sup&gt;</td></tr><tr><td>Bone marrow derived macrophages prepared from C57B/6 female mice</td><td></td><td></td><td>&lt;sup&gt;21&lt;/sup&gt;</td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;O, RNAse free</td><td>Thermo Scientific</td><td>10977-015</td><td>DEPC-treated water can be used</td></tr><tr><td>DMEM</td><td>Gibco</td><td>41965039</td><td></td></tr><tr><td>Glutamine</td><td>Gibco</td><td>25030081</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Gibco</td><td>11360-088</td><td></td></tr><tr><td>&amp;#946;-Mercaptoethanol</td><td>Gibco</td><td>31350010</td><td></td></tr><tr><td>Pen/Strep</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>Gentamicin</td><td>Sigma-Aldrich</td><td>G1397</td><td></td></tr><tr><td>FBS</td><td>Gibco</td><td>10270106</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline-PBS</td><td>Sigma-Aldrich</td><td>D8537</td><td></td></tr><tr><td>Brain heart infusion (BHI)</td><td>Merckmillipore</td><td>1104930500</td><td></td></tr><tr><td>Phenol saturated pH 4.3</td><td>Fisher</td><td>BP1751I-400</td><td></td></tr><tr><td>Chloroform</td><td>Fisher</td><td>BP1145-1</td><td></td></tr><tr><td>Iso-amyl alcohol</td><td>Sigma-Aldrich</td><td>W205702</td><td></td></tr><tr><td>Sodium acetate</td><td>Sigma-Aldrich</td><td>W302406</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>EDS</td><td></td></tr><tr><td>DNaseI</td><td>Fermentas</td><td>EN0521</td><td></td></tr><tr><td>SDS 10%</td><td>Sigma-Aldrich</td><td>L4522</td><td></td></tr><tr><td>Ethanol absolute</td><td>Merck Millipore</td><td>1070174000</td><td></td></tr><tr><td>37 &amp;deg;C, 5% CO&lt;sub&gt;2&lt;/sub&gt; forced-air incubator</td><td>Thermo Scientific</td><td>Model 3111</td><td></td></tr><tr><td>Cell scrapers</td><td>Nunc</td><td>179693</td><td></td></tr><tr><td>Kontes glass holder for 45 mm filters</td><td>Fisher</td><td>K953755-0045</td><td></td></tr><tr><td>MF-Millipore filters 45&amp;nbsp;mm, 0.45&amp;nbsp;&amp;micro;m</td><td>Merck Millipore</td><td>HAWP04700</td><td></td></tr><tr><td>SpeedVac system</td><td>Thermo Scientific</td><td>SPD131DDA</td><td></td></tr><tr><td>Vortex-Genie 2</td><td>Scientific Industries</td><td>Model G560E</td><td></td></tr><tr><td>NanoDrop</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>145 mm cell culture dishes</td><td>Greiner</td><td>639 160</td><td></td></tr><tr><td>1.7 ml tubes, RNase-free</td><td>Axygen</td><td>MCT-175-C</td><td></td></tr><tr><td>30 &amp;deg;C incubator</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>65 &amp;deg;C heat block</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>4 &amp;deg;C table centrifuge</td><td>Eppendorf</td><td>5417R</td><td></td></tr><tr><td>Sterile pipettes, 25 ml</td><td>Greiner</td><td></td><td></td></tr><tr><td>Falcon tubes, 50 ml</td><td>Greiner</td><td></td><td></td></tr><tr><td>Liquid nitrogen</td><td></td><td></td><td></td></tr></tbody></table>

rna purification from intracellularly grown listeria monocytogenes in macrophage cells

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage 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.

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...

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. ...

The overall goal of this procedure is to extract RNA from bacteria grown intracellularly in macrophage cells. This method can help answer key questions in the bacterial pathogenesis field, such as, what are the factors in genes that bacteria trigger in order to mediate infection? The main advantage of this technique is that it significantly enriches for bacterial RNA while minimizing contamination of host nucleic acids.Demonstrating the procedure will be Anna Pasechneck, a PhD student from my laboratory. To prepare cells for bacterial infection, on day one, use 145 millimeter dishes to seed two times ten to the seven bone marrow derived macrophage, or BMDM cells, in 30 milliliters of BMDM medium plus Pen/Strep. Incubate at 37 degrees Celsius and five percent carbon dioxide in a forced air incubator overnight.Inoculate a bacterial culture of wild type L.monocytogenes bacteria in 10 milliliters of brain heart infusion, or BHI, medium. Place tubes in an incubator in a slanted position and incubate at 30 degrees Celsius without shaking overnight. The following day, pre-warm BMDM medium without antibiotics and PBS to 37 degrees Celsius.Then use 25 milliliters of the warm PBS to wash the macrophage monolayer twice to remove the antibiotics before adding 30 milliliters of fresh BMDM medium to the cells. Next, wash the overnight L.monocytogenes culture by centrifuging 1.5 milliliters of the bacteria. Then use 1.5 milliliters of PBS to re-suspend the pellet.Then, using 0.5 milliliters of the washed bacterial suspension, infect each macrophage plate. If using multiple plates, space the infections 15 minutes apart to allow for harvesting each plate individually at the end of the infection. Following a 30 minutes incubation at 37 degrees Celsius, use PBS to wash the infected cells twice to remove unattached bacteria, and then add 30 milliliters of pre-warmed BMDM medium.Incubate for 30 minutes to allow bacteria to internalize. At one hour post infection, add gentamicin to a final concentration of 50 micrograms per milliliter to kill any remaining extracellular bacteria. Assemble the filter apparatus by placing a filter head on a collecting liquid flask with a vacuum outlet port.Then place a 0.45 micron filter on the filter head, followed by a cylinder funnel, and use a metal clamp to secure the different parts. At six hours post infection, examine the cell monolayer under the microscope before harvesting. Working with one plate at a time to harvest the bacteria, use PBS to wash the infected cells.Then, to lyse the macrophage cells, add 20 milliliters of ice cold RNAse free water and quickly but carefully use a cell scraper to scrape the cells off the plate. After collecting the cells into a 50 milliliter conical tube, vortex for 30 seconds, then centrifuge at 800 times g and four degrees Celsius for three minutes to remove macrophage cell nuclei. To collect bacteria, pass the supernatant through the filter using the vacuum system.Then, with tweezers, roll the filter and quickly transfer it to a 15 milliliter conical tube. Immediately use liquid nitrogen to snap freeze the tube. On the third day, prepare 400 microliters of a one to one mixture of acidified phenol and chloroform into separate 1.5 milliliter tubes for each sample.After mixing well, aspirate the top aqueous layer before adding 40 microliters of 10%SDS to the remaining solution. Thaw the tubes containing the filters on ice to keep them cold. Then to each tube, add 650 microliters of cold acetate EDTA or AE buffer.Working as quickly as possible, vigorously vortex the filter tube so that the filter whisks to the periphery of the tube and the buffer fully washes the filter. It may be necessary to invert the tube and vortex to fully wash off the bacteria from the filter. The most critical step of the procedure is to ensure the bacteria are efficiently removed from the filter.To do so, we repeat vortexing the tubes multiple times in different positions. Once the rest of the samples have been vortexed, briefly spin down the tubes. Next, after transferring the bacteria containing buffer from each tube to the tubes of SDS and phenol chloroform, place the tubes in a multi-tube vortex device and vortex at full speed for 10 minutes.Then, incubate the tubes in a heat block at 65 degrees Celsius for 10 minutes before spinning at maximum speed for five minutes. Transfer the aqueous layer to a fresh tube containing 40 microliters of three molar sodium acetate and one milliliter of 100%ethanol. After thoroughly vortexing the samples, incubate the tubes at minus 80 degrees Celsius for one hour or at minus 20 degrees Celsius overnight.Then, after centrifuging the samples, carefully aspirate ethanol from each tube before adding 500 microliters of cold 70%ethanol to each sample. After vortexing and spinning the samples again, carefully aspirate the ethanol from each tube and use a vacuum evaporator to dry the pellets for two minutes. Next, add 25 microliters of RNAse free water to each tube, and incubate at room temperature for 20 minutes.After vortexing and spinning down the samples, with a micro-volume UV/VIS spectrophotometer, measure the RNA concentration. Expect to extract about 0.5 to one microgram of nucleic acids per plate. To carry out Dnase treatment, set up the reactions in microcentrifuge tubes according to the text protocol.Incubate the samples at 37 degrees Celsius for 45 minutes. Then add 450 microliters of RNAse free water and 500 microliters of phenol chloroform IAA mix. Once the samples have been vortexed and spun at maximum speed for two minutes, transfer the aqueous layer to a new tube and add 500 microliters of chloroform IAA mix.After vortexing and spinning the samples again, transfer the aqueous layer to a new tube, before adding one milliliter of ethanol and 15 microliters of three molar sodium acetate. Following another vortex, incubate the tubes at minus 80 degrees Celsius for one hour or minus 20 degrees Celsius overnight. After the incubation, centrifuge at maximum speed and four degrees Celsius for 20 minutes.Carefully aspirate off the ethanol from each tube. Then add 500 microliters of cold 70%ethanol to each sample. After vortexing and spinning as before, carefully aspirate the ethanol from the tubes and use an unheated vacuum evaporator to dry the samples for two minutes without over drying.Then, add 12 microliters of RNAse free water, incubate at room temperature for two minutes, and vortex the samples before spinning them down. Immediately place the purified RNA samples on ice. Finally, use a micro-volume UV/VIS spectrophotometer to measure the RNA concentrations.Expect approximately 100 nanograms of RNA per combined samples. The Listeria RNA purified from macrophage cells is suitable for downstream transcription analysis by multiple available techniques. Shown here are the results of an RT qPCR experiment used to validate the transcription of known virulence genes during wild type L.monocytogenes growth in macrophages in comparison to exponential growth in the rich laboratory medium, BHI.In particular, the results of the hly and actA genes at six hours post infection are illustrated. Transcription levels were normalized to the levels of 16S ribosomal RNA as a reference gene. The data shown are representative of three independent biological repeats.Once mastered, this technique yields about 100 nanogram of total RNA, which is suitable for transcriptomic analysis by different methods, such as RT qPCR, microarray, and RNA-seq. While attempting this procedure, it is important to remember to work quickly as possible to avoid changes in gene transcription. While this protocol describes the purification of Listeria RNA, it can be easily modified to other bacterial pathogens.

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