Name: rna purification from intracellularly grown listeria monocytogenes in macrophage cells
Uploaded: 2016-06-04T14:00:00
Description: Watch this Scientific Journal Video about RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells at JoVE.com

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

Nota: Durante todo el experimento, las células de macrófagos se incubaron a 37 ° C en un incubador de CO2 de aire forzado 5% y sacados de la incubadora sólo para las manipulaciones experimentales, que se llevan a cabo en una cabina de seguridad biológica de Clase II. Trabajar con L. bacterias monocytogenes es de acuerdo al nivel de seguridad biológica 2 regulaciones. 1. Preparación de células y la infección bacteriana (Día 1 y 2) <ol><li> Día 1 <ol><li> Células de semil...

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrative+genomic+analysis+identifies+isoleucine+and+CodY+as+regulators+of+Listeria+monocytogenes+virulence.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Listeria+monocytogenes+multidrug+resistance+transporters+and+cyclic+di-AMP,+which+contribute+to+type+I+interferon+induction,+play+a+role+in+cell+wall+stress.">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. 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A., Auerbuch, V., Glomski, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cell+biology+of+Listeria+monocytogenes+infection:+the+intersection+of+bacterial+pathogenesis+and+cell-mediated+immunity.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Listeria+monocytogenes+-+from+saprophyte+to+intracellular+pathogen.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Mycobacterium+tuberculosis+RNAs+synthesized+in+response+to+phagocytosis+by+human+macrophages+by+selective+capture+of+transcribed+sequences+(SCOTS).">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Listeria+transcriptional+landscape+from+saprophytism+to+virulence.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+Adaptation+of+Mycobacterium+tuberculosis+within+Macrophages:+Insights+into+the+Phagosomal+Environment.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+sequencing-based+discovery+of+the+Chlamydia+trachomatis+transcriptome.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+whole+bacterial+pathogen+transcription+within+infected+hosts.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+RNA-seq+of+pathogen+and+host.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+insights+into+transcriptional+adaptation+of+intracellular+mycobacteria+by+microbe-enriched+dual+RNA+sequencing.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+3'Seq+using+deepSuperSAGE+uncovers+transcriptomes+of+interacting+Salmonella+enterica+Typhimurium+and+human+host+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granulocyte/macrophage+colony-stimulating+factor+is+expressed+and+secreted+in+cultures+of+murine+L929+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+widely+used+Listeria+monocytogenes+strains+EGD,+10403S,+and+EGD-e+highlights+genomic+variations+underlying+differences+in+pathogenicity.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+a+gamma-interferon+receptor+that+regulates+macrophage+tumoricidal+activity.">Evidence for a gamma-interferon receptor that regulates macrophage tumoricidal activity.</a> J Exp Med. 160, 55-74 (1984).</li></ol>

El protocolo descrito aquí representa un método optimizado para el aislamiento de ARN bacteriana de L. monocytogenes bacterias que crecen intracelularmente en células de macrófagos. Este protocolo se basa en la lisis diferencial de células e incluye dos pasos principales para el enriquecimiento de ARN bacteriano: sedimentación núcleos de macrófagos utilizando centrifugación y una rápida acumulación de bacterias por filtración. Estos pasos son seguidos por un procedimiento de extracción de ARN está...

Patógenos bacterianos intracelulares ─ bacterias causantes de enfermedades infecciosas y capaz de crecer y reproducirse dentro de las células del huésped humano ─ son un importante problema de salud en todo el mundo 5. Para invadir y replicarse dentro de una célula de mamífero, patógenos intracelulares han adquirido sofisticados mecanismos de virulencia y factores. Aunque estos mecanismos son fundamentales para la capacidad de causar una enfermedad, se sabe poco acerca de su regulación y dinámica. Desde los perfiles de expresión de genes de bacterias cultivadas en medio líquido no reflejan el entorno real dentro de células huésped, hay una necesidad creciente de transcriptoma análisis de bacterias cultivadas en sus nichos intracelulares. Estos análisis permitirán al desciframiento de adaptaciones específicas bacterianas provocadas por el anfitrión y le ayudará a identificar nuevas dianas para el diseño terapéutico. El análisis del transcriptoma de bacterias que crecen de forma intracelular es muy difícil, puesto que superan en número a ARN de mamífero ARN bacteriano por lopor lo menos diez veces. En este manuscrito, se describe un método experimental para aislar el ARN bacteriano de Listeria monocytogenes bacterias que crecen dentro de las células de macrófagos murinos. El ARN extraído se puede utilizar para estudiar las adaptaciones intracelulares y mecanismos de virulencia de las bacterias patógenas por diversas técnicas de análisis de la transcripción, tales como RT-PCR, el ARN-Seq, microarrays y otras tecnologías basadas en hibridación. L. monocytogenes es el agente causante de la listeriosis en humanos, una enfermedad con manifestaciones clínicas dirigidas principalmente a los individuos inmunocomprometidos, ancianos y mujeres embarazadas 6. Eso es un patógeno intracelular facultativo Gram-positiva que invade una amplia gama de células de mamíferos que se han utilizado durante décadas como modelo en las interacciones huésped-patógeno estudia 7. Tras la invasión, que reside inicialmente en una vacuola o fagosoma (en el caso de las células fagocíticas), de la que debe escapar en tque acogerá citosol de la célula con el fin de replicar. Varios factores de virulencia han demostrado mediar en el proceso de escape, principalmente la hemolisina de formación de poros, listeriolisina O (LLO) y dos fosfolipasas adicionales 8. En el citosol las bacterias utilizan el anfitrión maquinaria polimerización de la actina para impulsarse en los filamentos de actina dentro de la célula y propagarse de célula a célula (Figura 1). Todos los principales factores de virulencia de L. monocytogenes involucrados en la invasión, la supervivencia intracelular y la replicación, son activados por el regulador de la transcripción de virulencia principal, PrfA. 8-10. En la última década, varios estudios realizados por nosotros y otros han métodos para el análisis del transcriptoma de bacterias que crecen de forma intracelular aplicado con éxito dentro de las células anfitrionas 2,11-15. Dos enfoques principales se utilizan para separar el ARN bacteriano de host-RNA que se basan en: 1) el enriquecimiento selectivo de ARN bacteriana y 2) el aislamiento de ARN por difelisis celular rential. El primer enfoque se basa en la hibridación de sustracción de extractos de ARN total de las moléculas de ARN de mamífero (por ejemplo, utilizando kits disponibles en el comercio) o la captura selectiva de secuencias transcritas bacterianas (escoceses) 11. El segundo enfoque se basa en la lisis diferencial de bacterias y células huésped, en el que se lisan las células huésped mientras que las células bacterianas se mantienen intactos. Las células bacterianas se separan a continuación a partir del lisado de la célula huésped, generalmente por centrifugación, y el ARN se extrae usando técnicas estándar. El principal problema con este enfoque es que, junto con las bacterias intactas, también se aíslan células huésped núcleos, por lo tanto las preparaciones de ARN todavía contienen ARN de mamífero. Una forma de superar este problema es separar bacterias intactas de células huésped núcleos utilizando centrifugación diferencial, aunque este procedimiento por lo general toma tiempo el aumento de la preocupación de los cambios en el perfil de la expresión génica durante la extracción. En este artículo presentamos un ba mejorada y rápidaprotocolo de extracción de ARN cteria, que se basa en el enfoque de lisis diferencial de células. En primer lugar, L. monocytogenes células de macrófagos infectados se lisan con agua fría. A continuación, los núcleos de los macrófagos se eliminan mediante una breve centrifugación y bacterias intactas se recogen rápidamente en los filtros, de la que el ARN se aísla usando caliente extracción con fenol-SDS de los ácidos nucleicos bacterianos.

<table>
	<tbody>
		<tr>
			<td rowspan="2">Listeria monocytogenes 10403S</td>
			<td rowspan="2"></td>
			<td rowspan="2"></td>
			<td rowspan="2">20</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td rowspan="2">Bone marrow derived macrophages prepared from C57B/6 female mice</td>
			<td rowspan="2"></td>
			<td rowspan="2"></td>
			<td rowspan="2">21</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td>H2O, 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>b-Mercaptoethanol&#160;&#160;</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&#8217;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&#176;C, 5% CO2 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 mm, 0.45 &#181;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&#176;C incubator</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>65 &#176;C heat block</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4 &#176;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

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.

El sistema modelo se muestra en la Figura 1 e incluye células de macrófagos infectados con L. monocytogenes bacterias, que se replican en el citoplasma de los macrófagos. La figura 2 representa el esquema experimental. La figura 3 representa los resultados típicos de tales análisis de RT-qPCR de genes de virulencia durante WT L. monocytogenes en los macrófagos en comparación con el crecimiento en medio rico labor...

Watch this Scientific Journal Video about RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells at JoVE.com

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

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.

La purificación del ARN intracelularmente Grown<em&gt; Listeria monocytogenes</em&gt; En los macrófagos

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.

Research

JoVE Journal

Immunology and Infection

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

Los modelos de co-cultivo de Pseudomonas aeruginosa Biofilms Cultivado en las células vivas las vías respiratorias humanas

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living ...

Investigación

Inmunología e Infección

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

La medición de la carga bacteriana y la respuesta inmune en ratones infectados con Listeria monocytogenes</em

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells

Purificación por afinidad de los complejos de ribonucleoproteína Influenza Virus de la cromatina de las células infectadas

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the ...

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

La purificación del patógeno a partir de vacuolas<em&gt; Legionella</em&gt; Infectados por fagocitos

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe ...

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

La transmisión oral de<em&gt; Listeria monocytogenes</em&gt; En ratones a través de la ingestión de alimentos contaminados

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the ...

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Imaging InlC secreción de investigar la infección celular por el patógeno bacteriano<em&gt; Listeria monocytogenes</em

Bacterial intracellular  pathogens can be conceived as molecular tools to dissect cellular signaling  cascades due to their capacity to exquisitely ...

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Evaluación De La Invasión Bacteriana De Las Células Cardíacas En Cultivo Y Colonización Del Corazón En Ratones Infectados Usando Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised ...

Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-&#947; Responses

La infección experimental con<em&gt; Listeria monocytogenes</em&gt; Como un modelo para el estudio de host Respuestas interferón gamma

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Clasificación de células activadas por fluorescencia para la purificación de células dendríticas plasmocitoide de la médula ósea de ratones

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Small RNA Transfection in Primary Human Th17 Cells by Next Generation Electroporation

Small RNA Transfección en células primarias Th17 humano por Next Generation electroporación

CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated ...