Este trabajo fue apoyado por el NIH subvención 1SC2GM092789-01A1 y MS fue un becario de investigación de NMSU Howard Hughes Medical Research Programs institución universitaria. El video fue dirigido y producido por Amy lanasa y coordinado por el Dr. Philip Lewis con el Creative Media Institute en NMSU.

Procedimiento 1. Mosquito Crianza <ol><li> Rear mosquito Anopheles gambiae cepa G3 en un insectario en 27,5 ° C con 80% de humedad y ciclo de 12:12 horas de luz / oscuridad. </li><li> Las larvas se alimentan con la comida del suelo gato con levadura de cerveza en una proporción 1:1. </li><li> Alimentar a los mosquitos adultos en sangre de ratones en el día 3 post-emergencia para la producción de huevos. </li></ol> 2. Disección intestino del mosquito <ol><li> Autoclave herramientas de disección y pinzas (diapositivas). </li><li> Reúne 50 mosquitos utilizando un aspirador y colocarlos en la cama de CO 2 de flujo (Ultimate Flypad). Enjuague un espécimen mosquito secuencialmente en tres placas de Petri que contienen 70% de etanol para limpiar la superficie del cuerpo del mosquito. </li><li> Colocar la muestra sobre un portaobjetos de vidrio bajo una estereomicroscopía. Retire el intestino. </li><li> Reúne 50 agallas por condición de aislamiento de ADN metagenómica. Reúne 50 agallas por condición metagenómica para el aislamiento de ARN. </li></ol&gt; 3. Aislamiento de ADN metagenómica Nota: ADN metagenómico se aisló utilizando el Meta-G-Nome DNA Isolation Kit (Biotecnologías Epicentre) con la modificación descrita a continuación. <ol><li> Coloque 50 tripas de mosquitos en 300 l de TE. Homogeneizar ellos por 1 min utilizando Bio-Gen PRO200 homogeneizador (PRO Scientific Inc, EE.UU.) a la velocidad de 2.000 rpm en hielo. </li><li> Añadir 2 l de solución de lisozima Ready-Lyse y 1 l de RNasa A a la suspensión celular. Mezclar por agitación e incubar a 37 ° C durante 30 min. </li><li> Añadir 300 l de solución de lisis Meta-(2X) y 1 l de proteinasa K al tubo. Mezcle por vortex. Brevemente pulso a centrifugar el tubo para asegurar que toda la solución se encuentra en la parte inferior del tubo, y se incuba a 65 ° C durante 15 min, se enfría a temperatura ambiente, luego colocar en hielo durante 3-5 min. </li><li> Añadir 350 l de reactivo de precipitación de proteínas MPC al tubo y se mezcla mediante agitación en vórtex vigorosamente durante 10 segundos. </li><li> Pellet tque los desechos por centrifugación durante 10 min a 12.000 xg a 4 ° C. </li><li> Transferir el sobrenadante a un limpio de 2 ml tubo, y desechar el sedimento. </li><li> Añadir 570 l de isopropanol al sobrenadante. Mezclar mediante veces invirtiendo el tubo varias. </li><li> Pellet el ADN por centrifugación durante 10 min a 12.000 xg a 4 ° C. Eliminar el isopropanol sin desalojar el sedimento de ADN. </li><li> Añadir 500 l de etanol al 75% para lavar el sedimento. Centrifugar durante 5 min a 12.000 xg a 4 ° C. Eliminar el etanol sin perturbar el sedimento de ADN. Aire secar el sedimento a temperatura ambiente durante 2 min. Nota: No se exceda en seco el precipitado de ADN. </li><li> Resuspender el sedimento de ADN en 50 l de tampón TE. </li><li> Validar el tamaño del ADN aislado en comparación con el ADN de control fosmid (40 kb; 100 ng / l) proporcionado en el kit, a través de electroforesis en gel en un gel de agarosa 1%. </li></ol> 4. Aislamiento de ARN total Nota: Limpie el área de trabajocon RNaseZap para minimizar la contaminación de ARNasa. Metagenomic ARN se aisló de muestras de intestino del mosquito utilizando el TriPure Isolation Reagent (Roche) con una modificación menor. <ol><li> Ponga 50 tripas de mosquitos en un tubo de 2 ml con 500 l de reactivo de aislamiento TriPure. Homogeneizar durante 1 min a velocidad de 2.000 rpm en hielo usando un homogeneizador. Deje reposar a temperatura ambiente durante 5 min para permitir la disociación de los complejos de nucleoproteínas. </li><li> Añadir 100 l de cloroformo y agitar durante 12 segundos, y dejar reposar a temperatura ambiente durante 2 min. </li><li> Centrifugar a 10.000 xg durante 10 min. Con cuidado, saque el tubo sin molestar fases separadas. </li><li> Transferir la fase acuosa a un nuevo tubo, añadir 250 l de isopropanol, se mezcla bien, y poner a -20 º C durante 20 min. </li><li> Centrifugar a 12.000 xg durante 10 min a 4 ° C para precipitar el ARN. </li><li> Descartar el sobrenadante sin desalojar pellet. Lavar el pellet con 750 l de etanol al 75%. </li><li> Pipetear el sobrenadante sin perturbar el sedimento. LaIR secar el tubo durante 2 min. </li><li> Resuspender el RNA con 30 l de agua libre de nucleasas. </li><li> Tratar el ARN total con DNasa a 37 ° C durante 20 min y, a continuación inactivar la DNasa mediante la incubación a 75 ° C durante 15 min. ARN precipitado con etanol y se resuspende ARN en 30 l de agua libre de nucleasas. </li><li> Determinar la cantidad de RNA total utilizando un NanoDrop. </li></ol> 5. Ribosomal RNA agotamiento Nota: El protocolo fue desarrollado en base a los métodos descritos anteriormente 12,13,15. <ol><li> La amplificación por PCR de fragmentos de genes ribosomales de RNA Este paso crea muestra específica de piscinas rRNA, que se utilizan como plantillas para la transcripción in vitro para producir anticuerpos anti-sentido ARN ribosómico (arRNA) sondas que son complementarias a rRNA en el total de la muestra de ARN. <ol><li> Diseñar y sintetizar conjuntos de cebadores para el ARNr fragmento de PCR (Tabla 1). </li><li> Realizar la PCR en 50l de reacción utilizando Taq ADN polimerasa (Qiagen) con 50 ng de DNA plantilla, 1 x tampón de PCR, 1,5 mM de Mg 2 Cl, 0,2 m con cebadores de 35 ciclos de desnaturalización a 94 ° C durante 10 seg, 15 seg de recocido a 40 ° C para amplicón bacteriana 23S, y 50 ° C para los amplicones de otros, y extensión a 72 ° C durante 1 min (extensión final a 72 ° C durante 5 min). </li><li> Ver productos de PCR en gel de agarosa al 1%. </li><li> Purificar los productos de PCR utilizando el kit de purificación de PCR QIAquick con elución en 50 l de tampón de elución. Cuantificar la concentración usando NanoDrop. </li></ol></li><li> La transcripción in vitro de sondas marcadas con biotina rRNA anti-sentido (arRNA). Nota: En este paso, las sondas específicas de la muestra de diferentes amplicones de ARNr se sintetizan usando el kit MEGAscript T7. <ol><li> Configurar una reacción de 20 l para cada amplicón muestra rRNA específico mezclando los ingredientes indicados a continuación (Tabla 2). Incubar durante una noche a 37 ° C. <strong&gt; Nota: por lo general los rendimientos de la reacción&gt; 50 microgramos de ARN. </li><li> Añadir 1 l de DNasa I (incluido en el kit) a la reacción y se incuba a 37 ° C durante 20 min para eliminar la plantilla de ADN. </li><li> Añadir 100 l de etanol 100% a la reacción, se centrifuga a 12.000 xg durante 10 min a 4 ° C para precipitar sintetizado arRNA. Desechar el sobrenadante y lavar el precipitado con 750 l de etanol al 75%. Resuspender las sondas de ARN de cada 50 l de agua libre de nucleasas. </li><li> Cuantificar la concentración de ARN utilizando un NanoDrop. Las sondas se almacena a -80 ° C. </li></ol></li><li> rRNA resta con biotina arRNA. Nota: Este paso emplea la arRNA biotinilado para hibridarse a ARNr en el total de la muestra de ARN. Los híbridos rRNA-arRNA son capturados por estreptavidina con perlas magnéticas recubiertas. El procedimiento se basa en el protocolo descrito en la referencia 13 con modificaciones menores. <ol><li> Reactivos <ol><li> Preparar 0,1 N NaOH, 20X cloruros de sodioide-citrato (SSC) y 1 X SSC con 20% de formamida. </li></ol></li><li> Hibridación <ol><li> Mezclar 1 RNA total de microgramos con 16S y 23S sondas (0,75 g cada una), 18S mosquitos y sondas 28S (0,75 g cada uno), 1 l inhibidor de RNasa, 2,5 l de 20X tampón SSC, formamida 10 l 100% en un tubo de 200 l PCR , añadir agua libre de nucleasa a 50 l. </li><li> Colocar el tubo de reacción en un termociclador e incubar a 70 ° C durante 5 minutos para abrir la estructura secundaria y la deceleración a 25 ° C usando incrementos de 5 º C por 1 min cada ° C para permitir la hibridación de sondas de rRNA y arRNA. </li></ol></li><li> La eliminación del rRNA <ol><li> Transferir 300 mu l perlas recubiertas con estreptavidina en un tubo de 1,7 ml. Colocar el tubo en un estante de separación magnética para inmovilizar las perlas. Eliminar el sobrenadante. Volver a suspender las bolas en 300 l de NaOH 0,1 N y mix también. Coloque el tubo de nuevo a la rejilla magnética y pipetear el sobrenadante. Volver a suspender las bolas en 300 l de 1X SSC, mezclar bien, inmovilizar el talón y pipetear el sobrenadante. Repita el lavado con 300 l de 1X SSC una vez más. </li><li> Haga 3 alícuotas de perlas, 100 mu l cada uno, en tubos de 1,7 ml. Se coloca el tubo en la gradilla magnética, y eliminar el sobrenadante por pipeteo. Nota: Las alícuotas de perlas se utiliza durante 3 rondas de captura biotinilada rRNA arRNA híbridos. </li><li> Añadir 50 l SSC 1X con 20% de formamida en la reacción de hibridación 50 l. Transferir la reacción en el tubo alícuota talón primero, y mezclar bien. Incubar a TA durante 10 min para permitir que los biotinilados rRNA sonda-híbridos para ser capturado por perlas de estreptavidina. Ponga el tubo cada 2 minutos para mezclar durante la incubación. </li><li> Se coloca el tubo en la gradilla magnética para inmovilizar las perlas, transferir el sobrenadante a la segunda Aliquot de perlas, se mezcla bien y se incuba como anteriormente. Llevar a cabo la captura tercero mediante la transferencia de sobrenadante en el tubo tercio de perlas. Mezclar e incubar. </li><li> Transferencia no rRNA sobrenadante a un tubo nuevo y purificación conducta usando el kit RNeasy MinElute para eliminar formamida siguiendo el manual del fabricante. </li><li> Comprobar la eficacia agotamiento rRNA utilizando un Agilent 6000 RNA kit Pico chip en un Bioanalyzer (Figura 4). </li></ol></li></ol></li></ol> Nota: Los rRNA depleción de las muestras de ARN están sujetos a ARNm de amplificación si es necesario 8.

<ol>
	<li>Wang, Z., Gerstein, M., Snyder, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-Seq:+a+revolutionary+tool+for+transcriptomics.">RNA-Seq: a revolutionary tool for transcriptomics.</a> Nat. Rev. Genet. 10, 57-63 (2009).</li><li>Xie, W., 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=Pyrosequencing+the+Bemisia+tabaci+Transcriptome+Reveals+a+Highly+Diverse+Bacterial+Community+and+a+Robust+System+for+Insecticide+Resistance.">Pyrosequencing the Bemisia tabaci Transcriptome Reveals a Highly Diverse Bacterial Community and a Robust System for Insecticide Resistance.</a> PLoS One. 7, e35181 (2012).</li><li>Xie, L., 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=Profiling+the+metatranscriptome+of+the+protistan+community+in+Coptotermes+formosanus+with+emphasis+on+the+lignocellulolytic+system.">Profiling the metatranscriptome of the protistan community in Coptotermes formosanus with emphasis on the lignocellulolytic system.</a> Genomics. 99, 246-255 (2012).</li><li>Gosalbes, M. J., 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=Metatranscriptomic+approach+to+analyze+the+functional+human+gut+microbiota.">Metatranscriptomic approach to analyze the functional human gut microbiota.</a> PLoS One. 6, e17447 (2011).</li><li>Urich, T., 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=Simultaneous+assessment+of+soil+microbial+community+structure+and+function+through+analysis+of+the+meta-transcriptome.">Simultaneous assessment of soil microbial community structure and function through analysis of the meta-transcriptome.</a> PLoS One. 3, e2527 (2008).</li><li>Gilbert, J. 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=Detection+of+large+numbers+of+novel+sequences+in+the+metatranscriptomes+of+complex+marine+microbial+communities.">Detection of large numbers of novel sequences in the metatranscriptomes of complex marine microbial communities.</a> PLoS One. 3, e3042 (2008).</li><li>Gifford, S. M., Sharma, S., Rinta-Kanto, J. M., Moran, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+a+deeply+sequenced+marine+microbial+metatranscriptome.">Quantitative analysis of a deeply sequenced marine microbial metatranscriptome.</a> ISME J. 5, 461-472 (2011).</li><li>Poretsky, R. S., 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=Comparative+day/night+metatranscriptomic+analysis+of+microbial+communities+in+the+North+Pacific+subtropical+gyre.">Comparative day/night metatranscriptomic analysis of microbial communities in the North Pacific subtropical gyre.</a> Environmental microbiology. 11, 1358-1375 (2009).</li><li>Shrestha, P. M., Kube, M., Reinhardt, R., Liesack, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+activity+of+paddy+soil+bacterial+communities.">Transcriptional activity of paddy soil bacterial communities.</a> Environmental Microbiology. 11, 960-970 (2009).</li><li>Mettel, C., Kim, Y., Shrestha, P. M., Liesack, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+of+mRNA+from+soil.">Extraction of mRNA from soil.</a> Applied and Environmental Microbiology. 76, 5995-6000 (2010).</li><li>He, S., 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=Validation+of+two+ribosomal+RNA+removal+methods+for+microbial+metatranscriptomics.">Validation of two ribosomal RNA removal methods for microbial metatranscriptomics.</a> Nat. Meth. 7, 807-812 (2010).</li><li>Pang, X., 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=Bacterial+mRNA+purification+by+magnetic+capture-hybridization+method.">Bacterial mRNA purification by magnetic capture-hybridization method.</a> Microbiol. Immunol. 48, 91-96 (2004).</li><li>Stewart, F. J., Ottesen, E. A., DeLong, E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+quantitative+analyses+of+a+universal+rRNA-subtraction+protocol+for+microbial+metatranscriptomics.">Development and quantitative analyses of a universal rRNA-subtraction protocol for microbial metatranscriptomics.</a> ISME J. 4, 896-907 (2010).</li><li>Wang, Y., Gilbreath, T. M., Kukutla, P., Yan, G., Xu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+gut+microbiome+across+life+history+of+the+malaria+mosquito+Anopheles+gambiae+in+Kenya.">Dynamic gut microbiome across life history of the malaria mosquito Anopheles gambiae in Kenya.</a> PLoS One. 6, e24767 (2011).</li><li>Su, C., Sordillo, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+to+enrich+mRNA+from+total+prokaryotic+RNA.">A simple method to enrich mRNA from total prokaryotic RNA.</a> Mol. Biotechnol. 10, 83-85 (1998).</li><li>Gusmao, D. S., 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=Culture-dependent+and+culture-independent+characterization+of+microorganisms+associated+with+Aedes+aegypti+(Diptera:+Culicidae)+(L.)+and+dynamics+of+bacterial+colonization+in+the+midgut.">Culture-dependent and culture-independent characterization of microorganisms associated with Aedes aegypti (Diptera: Culicidae) (L.) and dynamics of bacterial colonization in the midgut.</a> Acta Trop. 115, 275-281 (2010).</li><li>Lindh, J. M., Terenius, O., Faye, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=16S+rRNA+gene-based+identification+of+midgut+bacteria+from+field-caught+Anopheles+gambiae+sensu+lato+and+A.+funestus+mosquitoes+reveals+new+species+related+to+known+insect+symbionts.">16S rRNA gene-based identification of midgut bacteria from field-caught Anopheles gambiae sensu lato and A. funestus mosquitoes reveals new species related to known insect symbionts.</a> Appl. Environ. Microbiol. 71, 7217-7223 (2005).</li><li>Simon, C., Daniel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metagenomic+analyses:+past+and+future+trends.">Metagenomic analyses: past and future trends.</a> Appl. Environ. Microbiol. 77, 1153-1161 (2011).</li><li>Wang, Y., Qian, P. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conservative+fragments+in+bacterial+16S+rRNA+genes+and+primer+design+for+16S+ribosomal+DNA+amplicons+in+metagenomic+studies.">Conservative fragments in bacterial 16S rRNA genes and primer design for 16S ribosomal DNA amplicons in metagenomic studies.</a> PLoS One. 4, e7401 (2009).</li><li>Hunt, D. E., 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=Evaluation+of+23S+rRNA+PCR+primers+for+use+in+phylogenetic+studies+of+bacterial+diversity.">Evaluation of 23S rRNA PCR primers for use in phylogenetic studies of bacterial diversity.</a> Appl. Environ. Microbiol. 72, 2221-2225 (2006).</li></ol>

Los autores declaran que no tienen intereses en conflicto financieros.

Una comunidad microbiana compleja reside en el ecosistema intestinal mosquito 14,16,17. Metatranscriptomic secuenciación (RNA-seq) puede revelar información de contexto funcional dependiente interrogando a todo el transcriptoma 4,18 microbiana. Técnicamente, oligo-d (T) el enriquecimiento de ARNm mediada procariota no es aplicable debido a la ausencia de de poli (A) las colas de los mensajeros. Alternativamente, el agotamiento de rRNA se ha utilizado para el enriquecimiento del ARNm. Aquí, hemo...

La próxima generación de la tecnología de secuenciación ha avanzado mucho estudio metagenómica, permitiendo evaluar la composición taxonómica y la funcionalidad de un conjunto genético microbiano. La secuenciación de ARN (RNA-Seq) 1 puede pasar por alto métodos de cultivo para investigar metatranscriptomes microbianos en el 2-5 contextos diferentes. Un obstáculo importante para microbiana RNA-seq es la dificultad en el enriquecimiento de ARNm, como las especies de ARNm procariótico no están establemente poliadenilado. Por lo tanto, oligo d (T) mediada por enriquecimiento mensajero no es aplicable. La eliminación del rRNA abundante es un enfoque alternativo para enriquecer ARNm. Los kits comerciales para el agotamiento de rRNA, como Microexpress kit bacteriana Enriquecimiento ARNm (Ambion), RiboMinus Transcriptome Isolation Kit (bacterias) (Life Technologies), y el ARNm de SÓLO kit de aislamiento de mRNA procariótico (Epicentre) que preferentemente degrada rRNA con una exonucleasa, se han utilizado para retirar rRNA 6-8. Sin embargo, las sondas de captura en Microexpresso RiboMinus son buenos para quitar rRNA conocido desde los típicos bacterias Gram-positivas y Gram-negativas-(ver especificaciones de los fabricantes), pero menos compatible con rRNA de microbios desconocidos. Por consiguiente, la supresión puede ser menos eficiente para las muestras 8-10 metagenómicas. Además, la fidelidad mRNA abundancia era cuestionable cuando el tratamiento se aplicó exonucleasa 11. En general, resta basado agotamiento rRNA fue menos sesgada y más eficaz en el enriquecimiento de mRNA en entornos metagenómicas 10-13. El intestino del mosquito se adapta a una comunidad microbiana dinámica 14. Estamos interesados ​​en la caracterización de la función del microbioma intestinal mosquito mediante el uso de RNA-Seq. En una muestra de ARN aislado de las entrañas de mosquitos, tanto de ARN de mosquitos y microbianas presentes. Aquí se describe un protocolo modificado para usar sondas específicas de la comunidad rRNA para agotar de manera eficiente rRNA mosquito y microbiana por hibridación sustractiva. El ARNm resultantemuestras enriquecidas son apropiados para RNA-Seq. El flujo de trabajo general se representa en la figura 1.

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagent/Material </td>
 </tr>
 <tr>
 <td>Meta-G-Nome 				DNA isolation kit</td>
 <td>Epicentre</td>
 <td>MGN0910</td>
 <td>Metagenomic 				DNA isolation</td>
 </tr>
 <tr>
 <td>TriPure </td>
 <td>Roche</td>
 <td>11667165001</td>
 <td>Mdetagenomic 				RNA isolation</td>
 </tr>
 <tr>
 <td>MEGAscript 				T7 kit</td>
 <td>Ambion</td>
 <td>AM1334</td>
 <td>In 				vitro synthesis of RNA probes</td>
 </tr>
 <tr>
 <td>RNaseZap</td>
 <td>Ambion</td>
 <td>AM9780</td>
 <td>RNase 				free working area </td>
 </tr>
 <tr>
 <td>Biotin-16-UTP</td>
 <td>Roche</td>
 <td>11388908910</td>
 <td>In 				vitro synthesis of RNA</td>
 </tr>
 <tr>
 <td>Biotin-11-CTP</td>
 <td>Roche</td>
 <td>4739205001</td>
 <td>In 				vitro synthesis of RNA</td>
 </tr>
 <tr>
 <td>Streptavidin 				magnetic beads</td>
 <td>NEB</td>
 <td>S1420S</td>
 <td>Capture 				of rRNA hybrids</td>
 </tr>
 <tr>
 <td>Magnetic 				separation rack</td>
 <td>NEB</td>
 <td>S1506S</td>
 <td>Capture 				of rRNA hybrids</td>
 </tr>
 <tr>
 <td>RNeasy 				mini kit</td>
 <td>QIAGEN</td>
 <td>74104</td>
 <td>Purification 				of subtracted RNA</td>
 </tr>
 <tr>
 <td>RNase-Free 				DNase Set</td>
 <td>QIAGEN</td>
 <td>79254</td>
 <td>Removal 				DNA contamination </td>
 </tr>
 <tr>
 <td>Agilent 				RNA 6000 Pico Kit</td>
 <td>Agilent 				Technologies Inc.</td>
 <td>5067-1513</td>
 <td>Electropherogram 				of RNA</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment </td>
 </tr>
 <tr>
 <td>Bio-Gen 				PRO200 				Homogenizer</td>
 <td>PRO 				Scientific</td>
 <td>01-01200</td>
 <td>Mosquito 				gut tissue homogenization</td>
 </tr>
 <tr>
 <td>NanoDrop 				1000 Spectrophotometer</td>
 <td>Thermo 				Scientific</td>
 <td></td>
 <td>DNA 				&amp; RNA quantitation</td>
 </tr>
 <tr>
 <td>2100 				Bioanalyzer</td>
 <td>Agilent 				Technologies Inc.</td>
 <td>G2940CA</td>
 <td>Electropherogram 				of RNA</td>
 </tr>
 </tbody>
</table>

depletion of ribosomal rna for mosquito gut metagenomic rna-seq

El intestino del mosquito acomoda las comunidades microbianas dinámicas a través de las diferentes etapas del ciclo de vida del insecto. Caracterización de la capacidad genética y la funcionalidad de la comunidad intestino proporcionará información sobre los efectos de la microbiota intestinal en los hábitos de vida de mosquitos. Metagenómica RNA-Seq se ha convertido en una herramienta importante para analizar transcriptomes de diversos microbios presentes en una comunidad microbiana. El ARN mensajero comprende normalmente sólo el 1-3% del total de ARN, mientras que rRNA constituye aproximadamente el 90%. Es un reto para enriquecer ARN mensajero a partir de una muestra de ARN microbiano metagenomic porque la mayoría de las especies de ARNm procariótico carecen de poli (A) las colas. Esto evita oligo d (T) de aislamiento de mRNA mediada. Aquí se describe un protocolo que emplea muestra derivada rRNA sondas de captura para eliminar el rRNA de una muestra metagenomic ARN total. Para empezar, tanto los mosquitos y microbianos fragmentos de subunidades pequeñas y grandes rRNA se amplificó a partir de ADN de una muestra comunitaria metagenómica. Entonces, la comunidadcomunitarias específicas sondas biotiniladas antisentido ARN ribosomal se sintetizan in vitro usando ARN polimerasa de T7. Las sondas biotiniladas rRNA se hibridan con el ARN total. Los híbridos son capturados por las perlas recubiertas con estreptavidina y se retira del ARN total. Este protocolo sustracción basado elimina eficazmente tanto mosquito y rRNA microbiana del total de la muestra de RNA. La muestra de ARNm enriquecido se procesa adicionalmente para la amplificación del ARN y el ARN Seq-.

El protocolo incluye tres secciones: (1) preparación de los moldes de ADN metagenómica para rRNA PCR, (2) la creación de muestreo específicos rRNA sondas de captura, (3) el agotamiento de rRNA de ARN total mediante hibridación sustractiva. Aislamiento de alta calidad metagenomic ADN y ARN es esencial para el proceso. El modificada Meta-G-Nome protocolo de aislamiento de ADN se obtiene de alta calidad de ADN metagenómico de tripas de mosquitos, como se muestra en la Figura 2. Puede ser difícil de ...

Watch this Scientific Journal Video about Depletion of Ribosomal RNA for Mosquito Gut Metagenomic RNA-seq at JoVE.com

Depletion of Ribosomal RNA for Mosquito Gut Metagenomic RNA-seq

Un ARN ribosómico (ARNr) de protocolo agotamiento fue desarrollado para enriquecer el ARN mensajero (ARNm) de ARN-ss de la metatranscriptome intestino del mosquito. Sondas de muestreo específicos rRNA, que fueron utilizados para extraer el rRNA mediante sustracción, se crea a partir del mosquito y sus microbios intestinales. Rendimiento del protocolo puede resultar en la eliminación de aproximadamente el 90-99% de rRNA.

El agotamiento de ARN ribosomal de metagenómica intestino del mosquito RNA-seq

The mosquito gut accommodates dynamic microbial communities across different stages of the insect's life cycle. Characterization of the genetic capacity ...

depletion-of-ribosomal-rna-for-mosquito-gut-metagenomic-rna-seq

Research

JoVE Journal

Biology

19.5K vistas.  New Mexico State University. Un ARN ribosómico (ARNr) de protocolo agotamiento fue desarrollado para enriquecer el ARN mensajero (ARNm) de ARN-ss de la metatranscriptome intestino del mosquito. Sondas de muestreo específicos rRNA, que fueron utilizados para extraer el rRNA mediante sustracción, se crea a partir del mosquito y sus microbios intestinales. Rendimiento del protocolo puede resultar en la eliminación de aproximadamente el 90-99% de rRNA.

Video: Depletion of Ribosomal RNA for Mosquito Gut Metagenomic RNA-seq

Large-Scale Screens of Metagenomic Libraries

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation. Generating a streamlined procedure for creating and screening metagenomic libraries is therefore useful for efficient high-throughput investigations into the genetic and metabolic properties of uncultured microbial assemblages. Here, key protocols are presented on video, which we propose is the most useful format for accurately describing a long process that alternately depends on robotic instrumentation and (human) manual interventions. First, we employed robotics to spot library clones onto high-density macroarray membranes, each of which can contain duplicate colonies from twenty-four 384-well library plates. Automation is essential for this procedure not only for accuracy and speed, but also due to the miniaturization of scale required to fit the large number of library clones into highly dense spatial arrangements. Once generated, we next demonstrated how the macroarray membranes can be screened for genes of interest using modified versions of standard protocols for probe labeling, membrane hybridization, and signal detection. We complemented the visual demonstration of these procedures with detailed written descriptions of the steps involved and the materials required, all of which are available online alongside the video.

A gran escala de pantallas de bibliotecas metagenómicas

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation.  Generating a ...

Investigación

Biología

Protocol for Mosquito Rearing (A. gambiae)

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night cycle. For this procedure, you'll need mosquito cages, 10% sterile sucrose solution, paper towels, beaker, whatman filter paper, glass feeders, human blood and serum, water bath, parafilm, distilled water, clean plastic trays, mosquito food (described below), mosquito net to cover the trays, vacuum, and a collection chamber to collect adults.

Protocol for Mosquito Rearing A. gambiae

This video illustrates the general techniques used to rear Anopheles gambiae in the laboratory. The methods for caring for laboratory mosquitoes are demonstrated through all stages of the organism's life cycle from larvae to pupae to blood-feeding adults.

Protocolo para la cría de los mosquitos (A. gambiae)

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night ...

Substrate Generation for Endonucleases of CRISPR/Cas Systems

The interaction of viruses and their prokaryotic hosts shaped the evolution of bacterial and archaeal life. Prokaryotes developed several strategies to evade viral attacks that include restriction modification, abortive infection and CRISPR/Cas systems. These adaptive immune systems found in many Bacteria and most Archaea consist of clustered regularly interspaced short palindromic repeat (CRISPR) sequences and a number of CRISPR associated (Cas) genes (Fig. 1) 1-3. Different sets of Cas proteins and repeats define at least three major divergent types of CRISPR/Cas systems 4. The universal proteins Cas1 and Cas2 are proposed to be involved in the uptake of viral DNA that will generate a new spacer element between two repeats at the 5' terminus of an extending CRISPR cluster 5. The entire cluster is transcribed into a precursor-crRNA containing all spacer and repeat sequences and is subsequently processed by an enzyme of the diverse Cas6 family into smaller crRNAs 6-8. These crRNAs consist of the spacer sequence flanked by a 5' terminal (8 nucleotides) and a 3' terminal tag derived from the repeat sequence 9. A repeated infection of the virus can now be blocked as the new crRNA will be directed by a Cas protein complex (Cascade) to the viral DNA and identify it as such via base complementarity10. Finally, for CRISPR/Cas type 1 systems, the nuclease Cas3 will destroy the detected invader DNA 11,12 .
These processes define CRISPR/Cas as an adaptive immune system of prokaryotes and opened a fascinating research field for the study of the involved Cas proteins. The function of many Cas proteins is still elusive and the causes for the apparent diversity of the CRISPR/Cas systems remain to be illuminated. Potential activities of most Cas proteins were predicted via detailed computational analyses. A major fraction of Cas proteins are either shown or proposed to function as endonucleases 4.
Here, we present methods to generate crRNAs and precursor-cRNAs for the study of Cas endoribonucleases. Different endonuclease assays require either short repeat sequences that can directly be synthesized as RNA oligonucleotides or longer crRNA and pre-crRNA sequences that are generated via in vitro T7 RNA polymerase run-off transcription. This methodology allows the incorporation of radioactive nucleotides for the generation of internally labeled endonuclease substrates and the creation of synthetic or mutant crRNAs. Cas6 endonuclease activity is utilized to mature pre-crRNAs into crRNAs with 5'-hydroxyl and a 2',3'-cyclic phosphate termini.

CRISPR/Cas systems mediate adaptive immunity in Bacteria and Archaea. Many Cas proteins are proposed to act as endoribonucleases acting on crRNA precursors of varying length. Here we illustrate three different approaches to generate pre-crRNA substrates for the biochemical analysis of Cas endonuclease activity.

Sustrato Generación para endonucleasas de CRISPR / Cas Sistemas

The  interaction of viruses and their prokaryotic hosts shaped the evolution of  bacterial and archaeal life. Prokaryotes developed several strategies to ...

RNA-seq Analysis of Transcriptomes in Thrombin-treated and Control Human Pulmonary Microvascular Endothelial Cells

The characterization of gene expression in cells via measurement of mRNA levels is a useful tool in determining how the transcriptional machinery of the cell is affected by external signals (e.g. drug treatment), or how cells differ between a healthy state and a diseased state. With the advent and continuous refinement of next-generation DNA sequencing technology, RNA-sequencing (RNA-seq) has become an increasingly popular method of transcriptome analysis to catalog all species of transcripts, to determine the transcriptional structure of all expressed genes and to quantify the changing expression levels of the total set of transcripts in a given cell, tissue or organism1,2 . RNA-seq is gradually replacing DNA microarrays as a preferred method for transcriptome analysis because it has the advantages of profiling a complete transcriptome, providing a digital type datum (copy number of any transcript) and not relying on any known genomic sequence3.
Here, we present a complete and detailed protocol to apply RNA-seq to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is based on our recent published study entitled "RNA-seq Reveals Novel Transcriptome of Genes and Their Isoforms in Human Pulmonary Microvascular Endothelial Cells Treated with Thrombin,"4 in which we successfully performed the first complete transcriptome analysis of human pulmonary microvascular endothelial cells treated with thrombin using RNA-seq. It yielded unprecedented resources for further experimentation to gain insights into molecular mechanisms underlying thrombin-mediated endothelial dysfunction in the pathogenesis of inflammatory conditions, cancer, diabetes, and coronary heart disease, and provides potential new leads for therapeutic targets to those diseases.
The descriptive text of this protocol is divided into four parts. The first part describes the treatment of human pulmonary microvascular endothelial cells with thrombin and RNA isolation, quality analysis and quantification. The second part describes library construction and sequencing. The third part describes the data analysis. The fourth part describes an RT-PCR validation assay. Representative results of several key steps are displayed. Useful tips or precautions to boost success in key steps are provided in the Discussion section. Although this protocol uses human pulmonary microvascular endothelial cells treated with thrombin, it can be generalized to profile transcriptomes in both mammalian and non-mammalian cells and in tissues treated with different stimuli or inhibitors, or to compare transcriptomes in cells or tissues between a healthy state and a disease state.

This protocol presents a complete and detailed procedure to apply RNA-seq, a powerful next-generation DNA sequencing technology, to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is generalizable to various cells or tissues affected by different reagents or disease states.

RNA-seq Análisis de transcriptomes en trombina tratamiento y de control pulmonar humana células endoteliales microvasculares

The characterization of gene expression in cells via measurement of mRNA levels is a useful tool in determining how the transcriptional machinery of the ...

Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons

One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the long-lasting gold standards is to sequence 16S ribosomal RNA (rRNA) gene amplicons generated by domain-level PCR reactions amplifying from genomic DNA. Traditionally, this was performed by cloning and Sanger (capillary electrophoresis) sequencing of PCR amplicons. The advent of next-generation sequencing has tremendously simplified and increased the sequencing depth for 16S rRNA gene sequencing. The introduction of benchtop sequencers now allows small labs to perform their 16S rRNA sequencing in-house in a matter of days. Here, an approach for 16S rRNA gene amplicon sequencing using a benchtop next-generation sequencer is detailed. The environmental DNA is first amplified by PCR using primers that contain sequencing adapters and barcodes. They are then coupled to spherical particles via emulsion PCR. The particles are loaded on a disposable chip and the chip is inserted in the sequencing machine after which the sequencing is performed. The sequences are retrieved in fastq format, filtered and the barcodes are used to establish the sample membership of the reads. The filtered and binned reads are then further analyzed using publically available tools. An example analysis where the reads were classified with a taxonomy-finding algorithm within the software package Mothur is given. The method outlined here is simple, inexpensive and straightforward and should help smaller labs to take advantage from the ongoing genomic revolution.

Characterizing microbial community has been a longstanding goal in environmental microbiology. Next-generation sequencing methods now allow for the characterization of microbial communities at an unprecedented depth with minimal cost and labor. We detail here our approach to sequence bacterial 16S ribosomal RNA genes using a benchtop sequencer.

La secuenciación de próxima generación de 16S ARN ribosomal genes Amplicones

One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the ...

An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, most notably developmental arrest and reduced metabolism. Global gene expression profiling via RNA-Seq can provide important insights into the transcriptional mechanisms of photoperiodic diapause. The Asian tiger mosquito, Aedes albopictus, is an outstanding organism for studying the transcriptional bases of diapause due to its ease of rearing, easily induced diapause, and the genomic resources available. This manuscript presents a general experimental workflow for identifying diapause-induced transcriptional differences in A. albopictus. Rearing techniques, conditions necessary to induce diapause and non-diapause development, methods to estimate percent diapause in a population, and RNA extraction and integrity assessment for mosquitoes are documented. A workflow to process RNA-Seq data from Illumina sequencers culminates in a list of differentially expressed genes. The representative results demonstrate that this protocol can be used to effectively identify genes differentially regulated at the transcriptional level in A. albopictus due to photoperiodic differences. With modest adjustments, this workflow can be readily adapted to study the transcriptional bases of diapause or other important life history traits in other mosquitoes.

An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

RNA-Seq analyses are becoming increasingly important for identifying the molecular underpinnings of adaptive traits in non-model organisms. Here, a protocol to identify differentially expressed genes between diapause and non-diapause Aedes albopictus mosquitoes is described, from mosquito rearing, to RNA sequencing and bioinformatics analyses of RNA-Seq data.

Un Experimental y Protocolo Bioinformática para análisis de ARN-ss de fotoperiódica diapausa en el Mosquito Tigre Asiático,<em&gt; Aedes albopictus</em

Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, ...

Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti

Electrophysiological recording of action potentials from sensory neurons of mosquitoes provides investigators a glimpse into the chemical perception of these disease vectors. We have recently identified a bitter sensing neuron in the labellum of female Aedes aegypti that responds to DEET and other repellents, as well as bitter quinine, through direct electrophysiological investigation. These gustatory receptor neuron responses prompted our sequencing of total mRNA from both male and female labella and tarsi samples to elucidate the putative chemoreception genes expressed in these contact chemoreception tissues. Samples of tarsi were divided into pro-, meso- and metathoracic subtypes for both sexes. We then validated our dataset by conducting qRT-PCR on the same tissue samples and used statistical methods to compare results between the two methods. Studies addressing molecular function may now target specific genes to determine those involved in repellent perception by mosquitoes. These receptor pathways may be used to screen novel repellents towards disruption of host-seeking behavior to curb the spread of harmful viruses.

Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti

Using two methods to estimate gene expression in the major gustatory appendages of Aedes aegypti, we have identified the set of genes putatively underlying the neuronal responses to bitter and repulsive compounds, as determined by electrophysiological examination.

Fisiológicos Grabaciones y RNA Secuenciación de los apéndices gustativas del mosquito de la fiebre amarilla<em&gt; Aedes aegypti</em

Electrophysiological recording of action potentials from sensory neurons of mosquitoes provides investigators a glimpse into the chemical perception of ...

Chitosan/Interfering RNA Nanoparticle Mediated Gene Silencing in Disease Vector Mosquito Larvae

Vector mosquitoes inflict more human suffering than any other organism&#8212;and kill more than one million people each year. The mosquito genome projects facilitated research in new facets of mosquito biology, including functional genetic studies in the primary African malaria vector Anopheles gambiae and the dengue and yellow fever vector Aedes aegypti. RNA interference- (RNAi-) mediated gene silencing has been used to target genes of interest in both of these disease vector mosquito species. Here, we describe a procedure for preparation of chitosan/interfering RNA nanoparticles that are combined with food and ingested by larvae. This technically straightforward, high-throughput, and relatively inexpensive methodology, which is compatible with long double stranded RNA (dsRNA) or small interfering RNA (siRNA) molecules, has been used for the successful knockdown of a number of different genes in A. gambiae and A. aegypti larvae. Following larval feedings, knockdown, which is verified through qRT-PCR or in situ hybridization, can persist at least through the late pupal stage. This methodology may be applicable to a wide variety of mosquito and other insect species, including agricultural pests, as well as other non-model organisms. In addition to its utility in the research laboratory, in the future, chitosan, an inexpensive, non-toxic and biodegradable polymer, could potentially be utilized in the field.

Here we describe a procedure for inhibiting gene function in disease vector mosquitoes through the use of chitosan/interfering RNA nanoparticles that are ingested by larvae.

El quitosano / ARN de interferencia mediada por nanopartículas silenciamiento génico en la enfermedad vectorial larvas de mosquitos

Vector mosquitoes inflict more human suffering than any other organism&#8212;and kill more than one million people each year. The mosquito genome projects ...

TChIP-Seq: Cell-Type-Specific Epigenome Profiling

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological functions have been extensively studied. Indeed, the advent of next-generation deep sequencing and chromatin immunoprecipitation (ChIP) via specific histone modification antibodies has revolutionized our view of epigenetic regulation across the genome. Conversely, tissues typically consist of diverse cell types, and their complex mixture poses analytic challenges to investigating the epigenome in a particular cell type. To address the cell type-specific chromatin state in a genome-wide manner, we recently developed tandem chromatin immunoprecipitation sequencing (tChIP-Seq), which is based on the selective purification of chromatin by tagged core histone proteins from cell types of interest, followed by ChIP-Seq. The goal of this protocol is the introduction of best practices of tChIP-Seq. This technique provides a versatile tool for tissue-specific epigenome investigation in diverse histone modifications and model organisms.

We describe a step-by-step protocol for tandem chromatin immunoprecipitation sequencing (tChIP-Seq) that enables the analysis of cell-type-specific genome-wide histone modification.

TChIP-Seq: Perfiles de celular-tipo-específico epigenoma

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological ...

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Quasi-metagenomics sequencing refers to the sequencing-based analysis of modified microbiomes of food and environmental samples. In this protocol, microbiome modification is designed to concentrate genomic DNA of a target foodborne pathogen contaminant to facilitate the detection and subtyping of the pathogen in a single workflow. Here, we explain and demonstrate the sample preparation steps for the quasi-metagenomics analysis of Salmonella enterica from representative food and environmental samples including alfalfa sprouts, ground black pepper, ground beef, chicken breast and environmental swabs. Samples are first subjected to the culture enrichment of Salmonella for a shortened and adjustable duration (4&#8211;24 h). Salmonella cells are then selectively captured from the enrichment culture by immunomagnetic separation (IMS). Finally, multiple displacement amplification (MDA) is performed to amplify DNA from IMS-captured cells. The DNA output of this protocol can be sequenced by high throughput sequencing platforms. An optional quantitative PCR analysis can be performed to replace sequencing for Salmonella detection or assess the concentration of Salmonella DNA before sequencing.

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Here, we present a protocol to prepare DNA samples from food and environmental microbiomes for concerted detection and subtyping of Salmonella through quasimetagenomic sequencing. The combined use of culture enrichment, immunomagnetic separation (IMS), and multiple displacement amplification (MDA) allows effective concentration of Salmonella genomic DNA from food and environmental samples.&#160;