Este trabalho foi financiado pelo NIH concessão 1SC2GM092789-01A1, e MS era um estudioso de pesquisa de NMSU Médico Howard Hughes Instituição Programas de Pesquisa de Graduação. O vídeo foi dirigido e produzido por Amy Lanasa e coordenado pelo Dr. Philip Lewis com o Creative Media Institute NMSU.

Procedimento 1. Elevando o mosquito <ol><li> Rear mosquito Anopheles gambiae estirpe G3 em insetário de 27,5 ° C com uma humidade de 80% e no ciclo de 12:12 horas luz / escuridão. </li><li> Alimentar as larvas com alimentos chão gato com levedura de cerveja na proporção de 1:1. </li><li> Alimentar os mosquitos adultos em sangue de camundongos no dia 3 pós-emergência para a produção de ovos. </li></ol> 2. Dissecção Gut mosquito <ol><li> Autoclave instrumentos de dissecação (slides e fórceps). </li><li> Colete 50 mosquitos usando um aspirador e colocá-los em CO cama fluxo 2 (Ultimate Flypad). Enxaguar um espécime mosquito sequencialmente em três placas de Petri contendo 70% de etanol para limpar a superfície do corpo do mosquito. </li><li> Coloque a amostra sobre uma lâmina de vidro sob uma lupa estereoscópica. Remover o intestino. </li><li> Colete 50 coragem por condição de isolamento de DNA metagenômico. Colete 50 coragem por condição de isolamento do RNA metagenomico. </li></ol&gt; 3. Isolamento de DNA metagenomico Nota: DNA metagenômico é isolado utilizando o Meta-G-Nome DNA Isolation Kit (Epicentre Biotechnologies), com a modificação descrita abaixo. <ol><li> Coloque 50 entranhas de mosquito em 300 TE ul. Homogeneizar durante 1 min, utilizando Bio-Gen PRO200 homogeneizador (PRO Scientific Inc, EUA) a 2000 rpm de velocidade sobre o gelo. </li><li> Adicionam-se 2 jil de Ready-Lyse solução de lisozima e 1 ul de ARNase A à suspensão de células. Mistura por agitação em vórtex e incubar a 37 ° C durante 30 min. </li><li> Adicionar 300 ul de Meta-Lysis Solution (2X) e 1 ml de Proteinase K para o tubo. Misture por vórtex. Resumidamente pulso-centrifugar o tubo para assegurar que a totalidade da solução está no fundo do tubo, e incubar a 65 ° C durante 15 minutos, arrefecer até à temperatura ambiente, e depois colocar em gelo durante 3-5 min. </li><li> Adicionar 350 ul de reagente de precipitação de proteínas MPC para o tubo e misturar vigorosamente em vortex durante 10 seg. </li><li> Pellet tele detritos por centrifugação durante 10 min a 12000 xg a 4 ° C. </li><li> Transferir o sobrenadante para um tubo limpo de 2 ml, e rejeitar o sedimento. </li><li> Adicionar 570 uL de isopropanol ao sobrenadante. Misture por vezes invertendo o tubo várias. </li><li> Pellet o DNA por centrifugação durante 10 min a 12000 xg a 4 ° C. Remover o isopropanol sem desalojar o sedimento de DNA. </li><li> Adicionar 500 ul de etanol a 75% para lavar o sedimento. Centrifugar durante 5 minutos a 12000 xg a 4 ° C. Remover o etanol, sem perturbar o sedimento de DNA. Ar-secar o sedimento à temperatura ambiente durante 2 min. Nota: Não excesso de seca, o pellet de DNA. </li><li> Ressuspender o sedimento de ADN em 50 ul de tampão TE. </li><li> Validar o tamanho do DNA isolado em comparação com o ADN de controlo Fosmid (40 kb, 100 ng / ul) fornecido no kit, através de electroforese em gel num gel de agarose a 1%. </li></ol> 4. Isolamento de RNA total Nota: Limpe a área de trabalhocom RNaseZap para minimizar a contaminação com RNase. RNA metagenoma foi isolado a partir de amostras intestinais de mosquitos usando o Reagente de Isolamento TriPure (Roche) com uma pequena modificação. <ol><li> Coloque 50 entranhas de mosquito em um tubo de 2 ml de reagente com 500 ul TriPure isolamento. Homogeneizar durante 1 min a 2000 rpm de velocidade em gelo usando um homogeneizador. Deixe sentar à temperatura ambiente durante 5 min para permitir que a dissociação de complexos nucleoproteicos. </li><li> Adicionam-se 100 ul de clorofórmio e agitar em vortex por 12 segundos, e deixe sentar à temperatura ambiente durante 2 min. </li><li> Centrifugar a 10.000 xg durante 10 min. Retire cuidadosamente o tubo sem perturbar fases separadas. </li><li> Transferir fase aquosa para um novo tubo, adicionar 250 uL de isopropanol, misturar bem e colocou-se a -20 ° C durante 20 min. </li><li> Centrifugar a 12.000 xg durante 10 min a 4 ° C para precipitar o RNA. </li><li> Desprezar o sobrenadante sem desalojar pelota. Lavar o sedimento com 750 ul de etanol a 75%. </li><li> Pipetar fora o sobrenadante sem perturbar o sedimento. Ari secar o tubo durante 2 min. </li><li> Ressuspender o RNA com 30 ul de água livre de nuclease. </li><li> Tratar o ARN total com ADNase a 37 ° C durante 20 min e, em seguida, desactivar a DNase por incubação a 75 ° C durante 15 min. RNA precipitado com etanol e RNA ressuspender em 30 ul de água livre de nuclease. </li><li> Determinar a quantidade de ARN total utilizando um NanoDrop. </li></ol> 5. Esgotamento RNA ribossomal Nota: O protocolo foi desenvolvido com base em métodos descritos anteriormente 12,13,15. <ol><li> A amplificação por PCR de fragmentos de genes de RNA ribossômico Este passo cria pools de amostras específicas do rRNA, que serão utilizadas como moldes para a transcrição in vitro para a produção de anti-sentido de RNA ribossomal (arRNA) sondas que são complementares ao rRNA na amostra de RNA total. <ol><li> Projetar e sintetizar conjuntos de primers para rRNA fragmento de PCR (Tabela 1). </li><li> Executar PCR em 50reacção ul usando DNA-polimerase Taq (Qiagen) com 50 ng de ADN molde, 1 x tampão de PCR, 1,5 mM Mg 2 Cl, 0,2 uM primários com 35 ciclos de desnaturação a 94 ° C durante 10 seg, 15 seg de recozimento a 40 ° C para amplicon 23S bacteriano, e 50 ° C para os amplicons outros, e que se estendem a 72 ° C durante 1 min (extensão final a 72 ° C durante 5 min). </li><li> Ver os produtos de PCR em gel de agarose a 1%. </li><li> Purifica-se os produtos de PCR utilizando o kit de purificação PCR QIAquick com eluição em 50 ul de tampão de eluição. Quantificar a concentração usando NanoDrop. </li></ol></li><li> Transcrição in vitro de sondas marcadas com biotina-anti-sentido de rRNA (arRNA). Nota: Neste passo, as sondas específicas de amostras de vários amplicons de rRNA são sintetizados utilizando o kit MEGAscript T7. <ol><li> Defina-se uma reacção de 20 uL de cada amostra de rRNA amplicon específico misturando os ingredientes a seguir (Tabela 2). Incubar durante a noite a 37 ° C. <strong&gt; Nota: normalmente os rendimentos de reação&gt; 50 ug de RNA. </li><li> Adicionar 1 ml de DNase I (incluso no kit) à reacção e incuba-se a 37 ° C durante 20 min para remover o molde de DNA. </li><li> Adicionam-se 100 ul de etanol a 100% para a reacção, de centrifugação a 12.000 xg durante 10 min a 4 ° C para precipitar sintetizado arRNA. Elimine o sobrenadante e lavar o sedimento com 750 ul de etanol a 75%. Ressuspender sondas de RNA em 50 ul de água livre de nuclease. </li><li> Quantificar RNA usando uma concentração NanoDrop. Sondas de armazenar a -80 ° C. </li></ol></li><li> rRNA subtração com arRNA biotinilado. Nota: Este passo utiliza o arRNA biotinilado para hibridar com rRNA na amostra de RNA total. Os híbridos rRNA-arRNA são capturados por estreptavidina-pérolas magnéticas revestidas. O procedimento baseia-se no protocolo descrito na referência 13, com pequenas modificações. <ol><li> Reagentes <ol><li> Prepare NaOH 0,1 N, 20X cloro sódioide-citrato (SSC) e 1 X SSC com formamida a 20%. </li></ol></li><li> Hibridização <ol><li> Misturar 1 ug de RNA total com 16S e 23S sondas (0,75 ug de cada), e sondas de mosquito 18S 28S (0,75 ug de cada), 1 ul de inibidor de RNase, 2,5 ul de tampão 20X SSC, 10 ul de formamida a 100% em um tubo de PCR 200 ul , adicione a água livre de nuclease a 50 ul. </li><li> Colocar o tubo de reacção num termociclador e incubar a 70 ° C durante 5 minutos, para abrir a estrutura secundária e rampa para baixo a 25 ° C, utilizando incrementos de 5 ° C durante 1 min cada ° C para permitir a hibridação das sondas de rRNA e arRNA. </li></ol></li><li> Remoção de rRNA <ol><li> Transferir 300 uL de estreptavidina pérolas revestidas dentro de um tubo ml 1.7. Colocar o tubo em uma cremalheira de separação magnética para imobilizar as contas. Remover o sobrenadante. Voltar a suspender as pérolas em 300 ul de NaOH 0,1 N e mix bem. Colocar o tubo de volta para a cremalheira magnético e pipetar fora o sobrenadante. Voltar a suspender as pérolas em 300 ul de SSC 1X, misturar bem, imobilizar as contas e pipetar fora o sobrenadante. Repetir a lavagem com 300 ul de 1X SSC mais uma vez. </li><li> Fazer 3 aliquotas de grânulos, 100 ul de cada, em 1,7 ml tubos. Colocar o tubo no suporte magnético, e remover o sobrenadante por pipetagem. Nota: As alíquotas de contas são utilizadas para a captura de três rodadas de biotinilados rRNA-arRNA híbridos. </li><li> Adicionar 50 ul de SSC 1X, com formamida a 20% da reacção de hibridação em 50 ul. Transferir a mistura reaccional para o tubo de primeira alíquota do grânulo, e misturar bem. Incubar à temperatura ambiente durante 10 min para permitir que os biotinilados sonda de rRNA-híbridos para ser capturado por esferas de estreptavidina. Flick o tubo a cada 2 minutos para misturar, durante a incubação. </li><li> Colocar o tubo no suporte magnético para imobilizar as contas, transferir o sobrenadante para o segundo aliquot dos grânulos, misturar bem e incubar como acima. Realizar a captura terceira transferindo sobrenadante para o terceiro tubo de esferas. Misturar e incubar. </li><li> Transferência não-rRNA sobrenadante para um novo tubo e a purificação utilizando o kit MinElute conduta RNeasy para remover formamida seguindo o manual do fabricante. </li><li> Verificar a eficiência depleção rRNA usando um Agilent 6000 kit RNA chip de Pico em um Bioanalyzer (Figura 4). </li></ol></li></ol></li></ol> Nota: As amostras de rRNA depleção de RNA são sujeitos a amplificação de mRNA se necessário 8.

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Os autores declaram que não têm interesses conflitantes financeiros.

A comunidade microbiana complexa reside no ecossistema estômago do mosquito 14,16,17. Metatranscriptomic seqüenciamento (RNA-seq) pode revelar informações dependente do contexto funcional interrogando o microbiana todo transcriptoma 4,18. Tecnicamente, oligo-d enriquecimento (T) de mRNA mediada procariótico não é aplicável devido à ausência de poli estáveis ​​(A) tails dos mensageiros. Alternativamente, a depleção de rRNA foi usado para o enriquecimento de mRNA. Aqui, foi desenvolv...

Próxima geração de tecnologia de seqüenciamento tem muito avançado estudo metagenômica, permitindo avaliar a composição taxonômica e funcionalidade genética de um conjunto microbiana. RNA-Sequencing (RNA-Seq) 1 pode ignorar os métodos baseados em cultura para investigar metatranscriptomes microbianas em 2-5 contextos diferentes. Um dos principais obstáculos à microbiana RNA-seq é a dificuldade no enriquecimento do mRNA, como as espécies de ARNm procariótico não são estavelmente polyadenylated. Portanto, oligo d (T) enriquecimento mensageiro mediada não é aplicável. Remoção de rRNA abundante é uma abordagem alternativa para o enriquecimento de mRNA. Kits comerciais de depleção de rRNA, tais como kit Enriquecimento bacteriana mRNA Microexpress (Ambion), RiboMinus Isolation Kit Transcriptoma (bactérias) (Life Technologies), e-mRNA Isolation kit APENAS Prokaryotic mRNA (Epicentre) que preferencialmente degrada rRNA com uma exonuclease, têm sido usados para a remoção de rRNA 6-8. No entanto, as sondas de captura em Microexpressou RiboMinus são bons para a remoção de rRNA a partir de conhecido típicos bactérias Gram-positivas e Gram-negativas (ver as especificações dos fabricantes), mas menos compatível com o rRNA de micróbios desconhecidos. Por conseguinte, a remoção pode ser menos eficiente para 8-10 metagenoma amostras. Além disso, a fidelidade abundância de ARNm era questionável, quando o tratamento com exonuclease foi aplicado 11. No geral, subtração baseada esgotamento rRNA foi menos tendencioso e mais eficaz no enriquecimento de mRNA em ambientes metagenômico 10-13. O intestino do mosquito acomoda uma comunidade microbiana dinâmica 14. Estamos interessados ​​em caracterizar a função do intestino microbiome mosquito usando RNA-Seq. Numa amostra de RNA isolado a partir dos intestinos de mosquitos, ambos os ARN de mosquito e microbianos estão presentes. Aqui, descrevemos um protocolo modificado para usar sondas específicas da comunidade rRNA de forma eficiente esgotar rRNA mosquito e microbiana por hibridização subtrativa. O mRNA resultanteamostras enriquecidas são apropriados para RNA-Seq. O fluxo de trabalho geral é mostrada na 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

O intestino do mosquito acomoda comunidades microbianas dinâmicas em diferentes estágios do ciclo de vida do inseto. Caracterização da capacidade genética e funcionalidade da comunidade intestino irá fornecer informações sobre os efeitos da microbiota intestinal em traços de vida do mosquito. Metagenomico RNA-Seq tornou-se uma importante ferramenta para analisar transcriptomas de micróbios vários presentes em uma comunidade microbiana. RNA mensageiro compreende geralmente apenas 1-3% de ARN total, enquanto rRNA constitui cerca de 90%. É um desafio para o enriquecimento de RNA mensageiro a partir de uma amostra de metagenômico microbiana RNA pois a maioria das espécies de ARNm procariótico falta estáveis ​​poli (A) tails. Isto evita que o oligo d (T) de isolamento de mRNA mediada. Aqui, nós descrevemos um protocolo que emprega amostra derivada de rRNA capturar sondas para remover rRNA a partir de uma amostra de RNA metagenômico total. Para começar, os dois fragmentos de mosquito e microbianas rRNA pequenas e grandes subunidades são amplificados a partir de uma amostra de DNA metagenômico comunidade. Então, a comunicaçãonitárias específicas biotinilados anti-sentido de sondas de RNA ribossomal são sintetizados in vitro usando T7 ARN-polimerase. As sondas biotiniladas rRNA são hibridizadas para o RNA total. Os híbridos são capturados por esferas revestidas com estreptavidina e removido a partir do ARN total. Este protocolo de subtração baseada remove eficientemente tanto mosquito e rRNA microbiano a partir da amostra de RNA total. O mRNA da amostra enriquecida é adicionalmente processado para o ARN e amplificação de RNA-Seq.

O protocolo inclui três secções: (1) a preparação de moldes de ADN para metagenoma rRNA PCR, (2) a criação de sondas de amostra de rRNA de captura específicos, (3) a depleção de rRNA a partir de ARN total por hibridação subtractiva. Isolamento de alta qualidade metagenômico ADN e ARN são essenciais para o processo. A modificação Meta-G-Nome protocolo de isolamento de ADN de alta qualidade produz DNA metagenômico de vísceras de mosquitos, conforme mostrado na Figura 2. Ele pode ser um d...

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

A ribosomal RNA protocolo de depleção (rRNA) foi desenvolvido para enriquecer o RNA mensageiro (mRNA) de RNA-seq do intestino metatranscriptome mosquito. Sondas de amostras específicas rRNA, que foram usados ​​para remover rRNA via subtração, foram criados a partir do mosquito e seus micróbios intestinais. Desempenho do protocolo pode resultar na remoção de aproximadamente 90-99% do rRNA.

Esgotamento de RNA ribossômico para metagenomico Gut 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 visualizações.  New Mexico State University. A ribosomal RNA protocolo de depleção (rRNA) foi desenvolvido para enriquecer o RNA mensageiro (mRNA) de RNA-seq do intestino metatranscriptome mosquito. Sondas de amostras específicas rRNA, que foram usados ​​para remover rRNA via subtração, foram criados a partir do mosquito e seus micróbios intestinais. Desempenho do protocolo pode resultar na remoção de aproximadamente 90-99% do rRNA.

Vídeo: 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.

Grande Escala Telas de Bibliotecas metagenomic

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

Biologia

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 a Criação Mosquito (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 ...

A High Throughput Screen for Biomining Cellulase Activity from Metagenomic Libraries

Cellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel production 1. Chemical methods to modify or degrade cellulose typically require strong acids and high temperatures. As such, enzymatic methods have become prominent in the bioconversion process. While the identification of active cellulases from bacterial and fungal isolates has been somewhat effective, the vast majority of microbes in nature resist laboratory cultivation. Environmental genomic, also known as metagenomic, screening approaches have great promise in bridging the cultivation gap in the search for novel bioconversion enzymes. Metagenomic screening approaches have successfully recovered novel cellulases from environments as varied as soils 2, buffalo rumen 3 and the termite hind-gut 4 using carboxymethylcellulose (CMC) agar plates stained with congo red dye (based on the method of Teather and Wood 5). However, the CMC method is limited in throughput, is not quantitative and manifests a low signal to noise ratio 6. Other methods have been reported 7,8 but each use an agar plate-based assay, which is undesirable for high-throughput screening of large insert genomic libraries. Here we present a solution-based screen for cellulase activity using a chromogenic dinitrophenol (DNP)-cellobioside substrate 9. Our library was cloned into the pCC1 copy control fosmid to increase assay sensitivity through copy number induction 10. The method uses one-pot chemistry in 384-well microplates with the final readout provided as an absorbance measurement. This readout is quantitative, sensitive and automated with a throughput of up to 100X 384-well plates per day using a liquid handler and plate reader with attached stacking system.

This protocol describes a high throughput screen for cellulolytic activity from a metagenomic library expressed in Escherichia coli. The screen is solution based and highly automated, and uses one-pot chemistry in 384 well microplates with the final readout as an absorbance measurement.

A tela de elevada capacidade para Biomining Atividade da celulase de Bibliotecas metagenomic

Cellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

Transcrição in vitro e nivelamento de Gaussia mRNA luciferase Seguido por HeLa transfecção celular

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

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.

Geração de substrato para Endonucleases 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 Análise de transcriptomas de trombina-tratados e controle humano pulmonar células endoteliais 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.

O sequenciamento de próxima geração de 16S RNA ribossomal 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 ...

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.

Uma Experimental e Bioinformática Protocolo de Análises de RNA-seq de fotoperiódicas diapausa no Tigre Asiático Mosquito,<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.

Fisiológicas Recordings e RNA Sequenciação dos gustativas Appendages do Mosquito-febre amarela<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.

A quitosana / RNA de interferência Nanoparticle Mediated silenciamento gênico na Doença Vector Mosquito Larvas

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