Recebemos assistência técnica da Biopolymers Facility da Harvard Medical School para bioanalisador. Este trabalho foi apoiado pela bolsa de pesquisa da National Multiple Sclerosis Society RG-1707-28516 (H.L.W. e S.L.).

Todos os métodos envolvendo animais de pesquisa descritos aqui foram aprovados pelo Comitê Institucional de Cuidado e Uso de Animais (IACUC) do Brigham and Women's Hospital, Harvard Medical School.
Todos os métodos que envolvem sujeitos de pesquisa humana descritos aqui estão de acordo com as diretrizes estabelecidas pelo Comitê de Pesquisa Humana dos Parceiros.
1. Coleta de amostras fecais
<ol><li>Autoclave ou prepare um tubo de microcentrífuga estéril e livre de nuclease de 2 mL com uma tampa de rosca para cada animal sujeito em um experimento.<ol><li>Para seres humanos, forneça um dispositivo de coleta de amostras de fezes apropriado, livre de nuclease e estéril para cada indivíduo.</li>
	</ol></li>
	<li>Coletar 25-100 mg (aproximadamente 1-4 pellets fecais para amostras fecais de camundongos) de amostras fecais de cada animal em um ambiente estéril. NOTA: Dois ou mais pellets fecais (~ 50 mg ou mais pesados) são preferidos para a obtenção de RNA da mais alta pureza.<ol><li>Utilize todos os Equipamentos de Proteção Individual (EPI) e materiais necessários, por exemplo: um par de luvas de laboratório, um spray desinfetante e uma toalha de papel estéril, para esterilizar a área de trabalho, onde o sujeito da pesquisa animal é colocado, para evitar a contaminação da amostra fecal.<ol><li>Para seres humanos, instrua cada sujeito de pesquisa / coletor a coletar 100-200 mg de amostras de fezes em um ambiente o mais estéril possível. Use a operação estéril padrão e evite a contaminação da amostra de fezes.</li>
		</ol></li>
	</ol></li>
	<li>Coletar amostras fecais de cada animal diretamente em um tubo de microcentrífuga de 2 mL com uma tampa de rosca sem tocar em quaisquer outras superfícies para evitar a contaminação.<ol><li>Para seres humanos, instrua cada sujeito a defecar diretamente em um dispositivo de coleta aplicável fornecido (por exemplo, um kit de coleta de amostras de fezes estéreis) para evitar a contaminação. NOTA: Instrua o sujeito a evitar a contaminação por superfícies de vaso sanitário, água, urina ou quaisquer outras superfícies/objetos não estéreis.</li>
	</ol></li>
	<li>Congelar amostras fecais coletadas em tubos microcentrífugos de 2 mL imediatamente a -80 °C ou colocar em um balde de gelo seco para a melhor quantidade e qualidade de RNA antes da ressuspensão das fezes, conforme descrito nas etapas abaixo.<ol><li>Para os espécimes de fezes humanas, alíquota de cada espécime fresco de 200 mg em tubos de microcentrífuga de 2 ml com tampas de rosca e congelá-los a -80 °C antes da ressuspensão das fezes, conforme descrito abaixo.<ol><li>Para o armazenamento de amostras de fezes não em tubos de microcentrífuga de 2 mL com tampas de rosca, pesar e transferir 100-200 mg cada uma das amostras congeladas em tubos microcentrífugas separados de 2 mL com tampas de rosca antes da ressuspensão das fezes, conforme descrito abaixo. NOTA: Para amostras de fezes humanas, evite tomar mais de 200 mg de amostras de fezes para isolamento de ARN, uma vez que pode causar dificuldades nos passos seguintes. Com uma amostra sobrecarregada em um tubo de microcentrífuga de 2 mL, a fase aquosa, a fase orgânica e a interfase podem não ser claramente formadas e separadas. O procedimento pode ser pausado aqui.</li>
		</ol></li>
	</ol></li>
</ol>2. Preparações de soluções de lavagem
<ol><li>Adicione 21 mL de etanol 100% grau da American Chemical Society (ACS) à Solução de Lavagem 1 fornecida no Kit de Isolamento de miRNA (consulte Tabela de Materiais) para atingir o volume final de 30 mL, conforme mostrado na garrafa. Vórtice até que tudo se dissolva na garrafa.</li>
	<li>Adicionar 40 mL de etanol 100% grau ACS à Solução de Lavagem 2/3 fornecida para atingir o volume final de 50 mL, conforme mostrado no frasco. Vórtice por 5 s ou até que a mistura final esteja bem misturada.</li>
</ol>3. Preparações de equipamentos e materiais
<ol><li>Pulverize a área de trabalho e o equipamento, por exemplo: a bancada de laboratório, a área de trabalho no exaustor químico e os racks de tubos de microcentrífuga, com uma solução de descontaminação de ribonuclease (RNase) (por exemplo, solução de descontaminação de RNase comercialmente disponível). Para evitar a contaminação, aplicar com a solução de descontaminação da RNase nas superfícies, onde e sempre que considerado necessário.</li>
	<li>Coloque um jaleco de laboratório limpo, coloque uma máscara facial e use luvas de laboratório apropriadas para proteger o RNA nas amostras fecais das nucleases presentes na pele humana. Pulverize luvas com uma solução de descontaminação de RNase e troque as luvas com frequência para evitar a contaminação.</li>
	<li>Prepare um balde de gelo seco para amostras fecais armazenadas a -80 °C para evitar o descongelamento antes da ressuspensão das fezes e um balde de gelo para materiais, por exemplo: Ácido-Fenol: Clorofórmio, para prolongar a vida útil. NOTA: Certifique-se de que os materiais, incluindo os meios utilizados no protocolo, são estéreis sem contaminação das nucleases.</li>
</ol>4. Resuspensão das fezes
<ol><li>Ressuspender 25-100 mg de amostras fecais em 600 μL de salina tamponada com fosfato (DPBS) estéril 1x Dulbecco. CUIDADO: As amostras fecais devem ser processadas imediatamente quando descongeladas a partir de -80 °C sem descongelamento parcial para minimizar a liberação de RNases e RNA celular à medida que os cristais de gelo se rompem nos compartimentos celulares internos e externos quando as células na amostra descongelam.<ol><li>Adicionar 600 μL de 1x DPBS ao tubo de microcentrífuga de 2 mL com uma tampa de rosca contendo amostras fecais à temperatura ambiente (RT).</li>
		<li>Incubar a mistura de amostras fecais submersas em 600 μL de 1x DPBS no tubo de microcentrífuga de 2 mL tampado por 30 min no RT.</li>
		<li>Ressuspenda a mistura por trituração com ponta de pipeta de 1 mL e vórtice bem com o tubo de microcentrífuga de 2 mL tampado. Para otimizar e aumentar a quantidade e a qualidade do RNA, ressuscite a mistura com um homogeneizador com a configuração para um ciclo a S4000 (ou 4000 rpm) e 45 s.</li>
	</ol></li>
</ol>5. Extração orgânica
CUIDADO: Use o exaustor químico perigoso para as seguintes etapas até a Etapa 6 com o uso de ácido-fenol: clorofórmio e etanol 100% grau ACS devido à sua toxicidade e inflamabilidade. Troque os EPIs conforme necessário e siga as devidas precauções padrão ao lidar com materiais perigosos.
<ol><li>Extrair ARN com 600 μL de ácido-fenol: clorofórmio (o volume de ácido-fenol: clorofórmio necessário é igual ao volume inicial de 1x DPBS adicionados no Passo 4.1).<ol><li>Adicionar 600 μL de ácido-fenol: clorofórmio à suspensão da etapa 4.1. NOTA: Retirar o ácido-fenol: clorofórmio da fase inferior do frasco, uma vez que a fase superior é misturada com um tampão aquoso. Se a interfase entre essas duas fases for perturbada, espere e retire o ácido-fenol: clorofórmio somente quando a interfase se restabelecer para evitar a contaminação.</li>
	</ol></li>
	<li>Vórtice a mistura por 60 s para misturar completamente. Alternativamente, para otimizar e aumentar a quantidade de RNA no rendimento, misture usando um homogeneizador com a configuração para um ciclo em S4000 e 45 s.</li>
	<li>Centrifugar por 15 min a 10.000 x g em RT para separar as fases aquosa e orgânica com uma microcentrífuga. Após a centrifugação, a interfase deve ser compacta. Caso contrário, repita a centrifugação. NOTA: Se a interfase não puder ser tão compacta quanto o desejado, possivelmente devido à proporção desigual do volume inicial com o volume de adição de ácido-fenol: clorofórmio após várias repetições de centrifugação, proceder à recuperação da fase aquosa com um maior cuidado para evitar a contaminação.</li>
	<li>Recupere a fase aquosa e transfira-a para um novo tubo de microcentrífuga de 2 mL com uma tampa de dobradiça (não fornecida pelo kit de isolamento de miRNA).<ol><li>Remova a fase aquosa (ou superior) cuidadosamente sem perturbar a fase inferior e transfira-a para um novo tubo de microcentrífuga de 2 mL com uma tampa de dobradiça. Observe o volume transferido (por exemplo, ~500 μL). NOTA: Quando a interfase é compacta e a fase superior é claramente separada, há possivelmente algumas minúsculas partículas residuais flutuando no topo da fase aquosa. Pipetar cuidadosamente para evitar estes resíduos e apenas recuperar a fase aquosa visível e claramente separada para garantir um rendimento de ARN de qualidade, mesmo que só possa obter um pequeno volume da fase aquosa.</li>
	</ol></li>
</ol>6. Isolamento final do RNA
<ol><li>Adicionar 1,25 volumes de etanol 100% grau RT ACS à fase aquosa no tubo de microcentrífuga de 2 mL (por exemplo, adicione 625 μL de etanol a 100% se 500 μL de fase aquosa for recuperado da Etapa 5.4.). Vórtice 3 s.</li>
	<li>Carregue a mistura aquosa de fase/etanol através do cartucho de filtro fornecido no kit de isolamento de miRNA.<ol><li>Para cada amostra, coloque o cartucho de filtro em um dos tubos de coleta fornecidos pelo kit.<ol><li>Pipetar e carregar 600 μL da mistura aquosa de fase/etanol no cartucho filtrante. NOTA: Vórtice a mistura brevemente para misturar completamente o etanol com fase aquosa antes de pipetar. Não mais de 700 μL da mistura aquosa de fase/etanol podem ser carregados de cada vez.</li>
		</ol></li>
		<li>Centrífuga a 10.000 x g por 90 s para filtrar através da mistura. Girar a uma velocidade mais alta pode danificar o filtro.</li>
		<li>Rejeitar o filtrado e repetir as etapas 6.2.1 a 6.2.2 até que toda a mistura seja filtrada através da mesma membrana filtrante em aplicações sucessivas. Mantenha e reutilize o mesmo tubo de coleta para lavar as etapas abaixo.</li>
		<li>Lavar o filtro com 700 μL de solução de lavagem de miRNA 1. CUIDADO: miRNA Wash Solution 1 contém tiocianato de guanidina que pode causar irritação da pele e danos oculares graves. Use os EPIs necessários, por exemplo: luvas, protetor facial, jaleco de laboratório de proteção. Troque as luvas com frequência, conforme necessário.<ol><li>Aplicar 700 μL de miRNA Wash Solution 1, a solução de trabalho preparada com o etanol 100% grau ACS, no cartucho filtrante.</li>
			<li>Centrífuga por 60 s para filtrar a Solução de Lavagem de miRNA 1 através do cartucho de filtro.</li>
			<li>Eliminar o filtrado do tubo de recolha e colocar o mesmo cartucho de filtro no mesmo tubo de recolha.</li>
		</ol></li>
		<li>Lave o filtro com solução de lavagem 2/3 uma vez cada com volumes de 700 μL, 500 μL e 250 μL consecutivamente.<ol><li>Aplicar 700 μL de Solução de Lavagem 2/3, a solução de trabalho preparada com o etanol 100% grau ACS, no cartucho filtrante.<ol><li>Centrífuga a 10.000 x g por 1 min.</li>
				<li>Eliminar o filtrado do tubo de recolha e colocar o mesmo cartucho de filtro no mesmo tubo de recolha.</li>
			</ol></li>
			<li>Aplicar 500 μL de solução de lavagem 2/3 no cartucho filtrante.<ol><li>Centrífuga a 10.000 x g por 1 min.</li>
				<li>Eliminar o filtrado do tubo de recolha e colocar o mesmo cartucho de filtro no mesmo tubo de recolha.</li>
			</ol></li>
			<li>Aplicar 250 μL de solução de lavagem 2/3 no cartucho do filtro.<ol><li>Centrífuga a 10.000 x g por 1 min.</li>
				<li>Eliminar o filtrado do tubo de recolha.</li>
			</ol></li>
			<li>Transfira o cartucho de filtro para um novo tubo de coleta e gire o conjunto por 5 minutos para remover o fluido residual do filtro.</li>
		</ol></li>
	</ol></li>
</ol>7. Elute RNA com 50 μL de água livre de nuclease
<ol><li>Transfira o cartucho de filtro para um novo tubo de coleta. Pipetar 50 μL de água isenta de nuclease para o centro do filtro e tampar o tubo de recolha.<ol><li>Incubar no RT por 10 min.</li>
		<li>Gire por 5 min a 8.000 x g para recuperar o RNA no novo tubo de coleta.</li>
		<li>Determinar a concentração e a pureza do RNA fecal recuperado usando um fluorômetro. O RNA fecal recuperado pode ser armazenado a -80 °C.</li>
	</ol></li>
</ol>

<ol>
	<li>Das, 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=The+extracellular+RNA+communication+consortium:+Establishing+foundational+knowledge+and+technologies+for+extracellular+RNA+research.">The extracellular RNA communication consortium: Establishing foundational knowledge and technologies for extracellular RNA research.</a> Cell. 177 (2), 231-242 (2019).</li><li>Ahmed, F. 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=Diagnostic+microRNA+markers+for+screening+sporadic+human+colon+cancer+and+active+ulcerative+colitis+in+stool+and+tissue.">Diagnostic microRNA markers for screening sporadic human colon cancer and active ulcerative colitis in stool and tissue.</a> Cancer Genomics &amp; Proteomics. 6 (5), 281-295 (2009).</li><li>Liu, 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=The+host+shapes+the+gut+microbiota+via+fecal+microRNA.">The host shapes the gut microbiota via fecal microRNA.</a> Cell Host &amp; Microbe. 19 (1), 32-43 (2016).</li><li>Viennois, 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=Host-derived+fecal+microRNAs+can+indicate+gut+microbiota+healthiness+and+ability+to+induce+inflammation.">Host-derived fecal microRNAs can indicate gut microbiota healthiness and ability to induce inflammation.</a> Theranostics. 9 (15), 4542-4557 (2019).</li><li>Liu, 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=Oral+administration+of+miR-30d+from+feces+of+MS+patients+suppresses+MS-like+symptoms+in+mice+by+expanding+Akkermansia+muciniphila.">Oral administration of miR-30d from feces of MS patients suppresses MS-like symptoms in mice by expanding Akkermansia muciniphila.</a> Cell Host &amp; Microbe. 26 (6), 779-794 (2019).</li><li>Masotti, 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=Quantification+of+small+non-coding+RNAs+allows+an+accurate+comparison+of+miRNA+expression+profiles.">Quantification of small non-coding RNAs allows an accurate comparison of miRNA expression profiles.</a> Journal of Biomedicine &amp; Biotechnology. 2009, 659028 (2009).</li><li>Green, M. R., Sambrook, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+win+the+battle+with+RNase.">How to win the battle with RNase.</a> Cold Spring Harbor Protocols. 2019 (2), 101857 (2019).</li><li>Franzosa, E. 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=Relating+the+metatranscriptome+and+metagenome+of+the+human+gut.">Relating the metatranscriptome and metagenome of the human gut.</a> Proceedings of the National Academy of Sciences. 111 (22), 2329-2338 (2014).</li><li>Wohnhaas, C. 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=Fecal+microRNAs+show+promise+as+noninvasive+Crohn's+disease+biomarkers.">Fecal microRNAs show promise as noninvasive Crohn's disease biomarkers.</a> Crohn's &amp; Colitis. 2 (1), 003 (2020).</li><li>Tomkovich, 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=Human+colon+mucosal+biofilms+and+murine+host+communicate+via+altered+mRNA+and+microRNA+expression+during+cancer.">Human colon mucosal biofilms and murine host communicate via altered mRNA and microRNA expression during cancer.</a> mSystems. 5 (1), (2020).</li><li>Tarallo, 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=Altered+fecal+small+RNA+profiles+in+colorectal+cancer+reflect+gut+microbiome+composition+in+stool+samples.">Altered fecal small RNA profiles in colorectal cancer reflect gut microbiome composition in stool samples.</a> mSystems. 4 (5), 70 (2019).</li><li>Xing, 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=Breed+differences+in+the+expression+levels+of+gga-miR-222a+in+laying+hens+influenced+H2S+production+by+regulating+methionine+synthase+genes+in+gut+bacteria.">Breed differences in the expression levels of gga-miR-222a in laying hens influenced H2S production by regulating methionine synthase genes in gut bacteria.</a> Research Square. , (2020).</li><li>Teng, Y., 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=Plant-derived+exosomal+microRNAs+shape+the+gut+microbiota.">Plant-derived exosomal microRNAs shape the gut microbiota.</a> Cell Host &amp; Microbe. 24 (5), 637-652 (2018).</li><li>Moloney, G. M., Viola, M. F., Hoban, A. E., Dinan, T. G., Cryan, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Faecal+microRNAs:+indicators+of+imbalance+at+the+host-microbe+interface.">Faecal microRNAs: indicators of imbalance at the host-microbe interface.</a> Beneficial Microbes. , 1-10 (2017).</li><li>Giannoukos, G., 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=Efficient+and+robust+RNA-seq+process+for+cultured+bacteria+and+complex+community+transcriptomes.">Efficient and robust RNA-seq process for cultured bacteria and complex community transcriptomes.</a> Genome Biology. 13 (3), 23 (2012).</li></ol>

Os autores declaram não haver interesses financeiros relevantes ou materiais relacionados ao método de pesquisa descrito neste artigo de protocolo.

É importante o uso da técnica livre de RNase para evitar a contaminação por RNase durante o isolamento7. Após a centrifugação e a formação de uma interfase compacta, é fundamental evitar a interfase, a fase inferior e o contaminante de partículas flutuando no topo da fase aquosa ao recuperar a fase aquosa. Além disso, duas etapas de lavagem com solução de lavagem 2/3 de 500 μL e 250 μL são adicionadas para eliminar contaminantes na membrana do filtro para uma qualidade otimizada. ...

O RNA extracelular está sendo reconhecido como um fator significativo que medeia muitos processos biológicos1. O RNA extracelular em fezes foi relatado pela primeira vez em 2008 como um marcador para câncer de cólon e colite ulcerativa ativa2, e foi recentemente revelado como um componente normal do lúmen intestinal e fezes e medeia as comunicações hospedeiro-micróbio 3,4,5. O objetivo deste protocolo de isolamento de RNA é extrair RNA extracelular de alta qualidade de amostras fecais coletadas de animais e seres humanos. O protocolo foi adaptado de um kit comercial de isolamento de miRNA. O RNA adquirido é utilizado para análises a jusante, como sequenciamento de RNA, RT-PCR e microarray. O protocolo inclui várias dicas importantes e úteis para maximizar a quantidade e a qualidade do RNA encontrado nas fezes de animais e humanos. A razão para desenvolver e otimizar este método de isolamento de RNA (incluindo microRNA) é diminuir o RNA microbiano nas fezes, limitar as variáveis nos estudos de pesquisa e analisar a composição de RNA no intestino sem contabilizar vários fatores de confusão e fontes de contaminação. De notar, este isolamento de RNA minimiza a liberação de RNA de células vivas e micróbios vivos (RNA celular). Ele se concentra em RNAs extracelulares que foram liberados pelas células intestinais ou foram adquiridos através da ingestão de alimentos. Principalmente, este método não é adequado para estudos em que o transcriptoma microbiano é investigado.

<table>
	<tbody>
		<tr>
			<td>Acid-Phenol: Chloroform, pH 4.5 (with IAA, 125:24:1)</td>
			<td>Thermo Fischer Scientific</td>
			<td>AM9720</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS, no calcium, no magnesium</td>
			<td>Thermo Fischer Scientific</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Gloves</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>mirVana miRNA Isolation Kit</td>
			<td>Thermo Fischer Scientific</td>
			<td>AM1561</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease-Free microcentrifuge tubes (1.5 mL, 2 mL)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease-Free Water (Not DEPC-treated)</td>
			<td>Thermo Fischer Scientific</td>
			<td>AM9937</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettor and Nuclease-Free Pipette tips (with filter)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PowerLyzer 24 Homogenizer</td>
			<td>QIAGEN</td>
			<td>13155</td>
			<td></td>
		</tr>
		<tr>
			<td>RNaseZap RNase Decontamination Solution</td>
			<td>Thermo Fischer Scientific</td>
			<td>AM9780</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex Shaker</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

fecal (micro) rna isolation

Está ficando claro que o RNA existe no lúmen intestinal e nas fezes em animais e humanos. O protocolo descrito abaixo isola o RNA total, incluindo microRNAs de amostras fecais de animais e seres humanos. O objetivo é isolar o RNA total com alta pureza e quantidade para análises a jusante, como sequenciamento de RNA, RT-PCR e microarray. As vantagens deste protocolo otimizado no isolamento de miRNA são as capacidades de isolar produtos de RNA altamente purificados com etapas de lavagem adicionais descritas, aumento da quantidade de RNA obtida com um método aprimorado na ressuspensão da amostra e importantes dicas de descontaminação. Uma limitação é a incapacidade de processar e purificar amostras maiores de mais de 200 mg, pois esses tamanhos de amostra causariam uma dificuldade na formação clara da interfase. Consequentemente, o grande tamanho da amostra pode contaminar a fase aquosa a ser extraída conforme descrito no protocolo com matérias orgânicas que afetam a qualidade do RNA isolado no final. No entanto, os isolados de ARN de uma amostra de até 200 mg são suficientes para a maioria das análises a jusante.

RNAs representativos foram isolados de 50 mg de amostras fecais de camundongos (2 pellets fecais de camundongos) e 100 mg de amostras de fezes humanas, respectivamente, e eluídos em água livre de nuclease de 50 μL. A análise espectrofotômetro da concentração sugere que uma quantidade total de 49 μg e 16 μg de RNA foram isolados, respectivamente (Tabela 1). A pureza do RNA foi alta, conforme indicado por uma relação A260/A280 de ~2,0 e uma relação A260/A230 de ~1,8 (Tabela 1)...

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Fecal micro RNA Isolation

Este protocolo isola RNA total de alta qualidade de amostras fecais de animais e seres humanos. Um kit comercial de isolamento de miRNA é usado com adaptação significativa para isolar o RNA puro com quantidade e qualidade otimizadas. Os isolados de RNA são bons para a maioria dos ensaios de RNA a jusante, como sequenciamento, micro-array e RT-PCR.

Isolamento de RNA fecal (micro)

It is becoming clear that RNA exists in the gut lumen and feces in animals and humans. The protocol described below isolates total RNA including microRNAs ...

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Unit 1

Fecal (micro) RNA Isolation

Diagnostic and Radiological Studies of the GI System

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4.5K visualizações.  Brigham and Women's Hospital and Harvard Medical School. Este protocolo isola RNA total de alta qualidade de amostras fecais de animais e seres humanos. Um kit comercial de isolamento de miRNA é usado com adaptação significativa para isolar o RNA puro com quantidade e qualidade otimizadas. Os isolados de RNA são bons para a maioria dos ensaios de RNA a jusante, como sequenciamento, micro-array e RT-PCR.

Vídeo: Fecal micro RNA Isolation

RNA Interference in Ticks

Ticks are obligate hematophagous ectoparasites of wild and domestic animals and humans, and are considered to be second worldwide to mosquitoes as vectors of human diseases1 and the most important vectors affecting cattle industry worldwide2. Ticks are classified in the subclass Acari, order Parasitiformes, suborder Ixodida and are distributed worldwide from Arctic to tropical regions3. Despite efforts to control tick infestations, these ectoparasites remain a serious problem for human and animal health4,5.
RNA interference (RNAi)6 is a nucleic acid-based reverse genetic approach that involves disruption of gene expression in order to determine gene function or its effect on a metabolic pathway. Small interfering RNAs (siRNAs) are the effector molecules of the RNAi pathway that is initiated by double-stranded RNA (dsRNA) and results in a potent sequence-specific degradation of cytoplasmic mRNAs containing the same sequence as the dsRNA trigger7-9. Post-transcriptional gene silencing mechanisms initiated by dsRNA have been discovered in all eukaryotes studied thus far, and RNAi has been rapidly developed in a variety of organisms as a tool for functional genomics studies and other applications10.
RNAi has become the most widely used gene-silencing technique in ticks and other organisms where alternative approaches for genetic manipulation are not available or are unreliable5,11. The genetic characterization of ticks has been limited until the recent application of RNAi12,13. In the short time that RNAi has been available, it has proved to be a valuable tool for studying tick gene function, the characterization of the tick-pathogen interface and the screening and characterization of tick protective antigens14. Herein, a method for RNAi through injection of dsRNA into unfed ticks is described. It is likely that the knowledge gained from this experimental approach will contribute markedly to the understanding of basic biological systems and the development of vaccines to control tick infestations and prevent transmission of tick-borne pathogens15-19.

A method for RNA interference (RNAi) by injection of dsRNA into unfed ticks is described. RNAi is the most widely used gene-silencing technique in ticks where the use of other methods of genetic manipulation has been limited.

RNA de interferência em Carrapatos

Ticks are obligate hematophagous ectoparasites of wild and domestic animals and humans, and are considered to be second worldwide to mosquitoes as vectors ...

Imunologia e Infecção

Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the generation of extreme population diversity that facilitates virus adaptation and evolution. Increasing evidence shows that the intrinsic error rates, and the resulting mutation frequencies, of RNA viruses can be modulated by subtle amino acid changes to the viral polymerase. Although biochemical assays exist for some viral RNA polymerases that permit quantitative measure of incorporation fidelity, here we describe a simple method of measuring mutation frequencies of RNA viruses that has proven to be as accurate as biochemical approaches in identifying fidelity altering mutations. The approach uses conventional virological and sequencing techniques that can be performed in most biology laboratories. Based on our experience with a number of different viruses, we have identified the key steps that must be optimized to increase the likelihood of isolating fidelity variants and generating data of statistical significance. The isolation and characterization of fidelity altering mutations can provide new insights into polymerase structure and function1-3. Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution4-7.

The present article describes the steps required to isolate and characterize RNA polymerase fidelity variants of RNA viruses and how to use mutation frequency data to confirm fidelity changes in tissue culture.

Isolamento de variantes de vírus de RNA Fidelity e Caracterização de frequência de mutação de vírus

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the ...

Development of Cell-type specific anti-HIV gp120 aptamers for siRNA delivery

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering (si)RNAs to inhibit the expression of complementary RNA transcripts is being exploited as a new class of therapeutics for a variety of diseases including HIV. Many previous reports have shown that novel RNAi-based anti-HIV/AIDS therapeutic strategies have considerable promise; however, a key obstacle to the successful therapeutic application and clinical translation of siRNAs is efficient delivery. Particularly, considering the safety and efficacy of RNAi-based therapeutics, it is highly desirable to develop a targeted intracellular siRNA delivery approach to specific cell populations or tissues. The HIV-1 gp120 protein, a glycoprotein envelope on the surface of HIV-1, plays an important role in viral entry into CD4 cells. The interaction of gp120 and CD4 that triggers HIV-1 entry and initiates cell fusion has been validated as a clinically relevant anti-viral strategy for drug discovery. 
Herein, we firstly discuss the selection and identification of 2'-F modified anti-HIV gp120 RNA aptamers. Using a conventional nitrocellulose filter SELEX method, several new aptamers with nanomolar affinity were isolated from a 50 random nt RNA library. In order to successfully obtain bound species with higher affinity, the selection stringency is carefully controlled by adjusting the conditions. The selected aptamers can specifically bind and be rapidly internalized into cells expressing the HIV-1 envelope protein. Additionally, the aptamers alone can neutralize HIV-1 infectivity. Based upon the best aptamer A-1, we also create a novel dual inhibitory function anti-gp120 aptamer-siRNA chimera in which both the aptamer and the siRNA portions have potent anti-HIV activities. Further, we utilize the gp120 aptamer-siRNA chimeras for cell-type specific delivery of the siRNA into HIV-1 infected cells. This dual function chimera shows considerable potential for combining various nucleic acid therapeutic agents (aptamer and siRNA) in suppressing HIV-1 infection, making the aptamer-siRNA chimeras attractive therapeutic candidates for patients failing highly active antiretroviral therapy (HAART).

Several 2&#x2019;-Fluoro RNA aptamers against HIV-1Ba-L gp120 with nanomole affinity are isolated from a RNA library by in vitro SELEX procedure. A new dual inhibitory function anti-gp120 aptamer-siRNA chimera is created and shows considerable promise for systemic anti-HIV therapy.

Desenvolvimento de células de tipo específico anti-HIV gp120 aptâmeros para entrega siRNA

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering ...

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with leukemia, severe burn wounds, or organ transplants1. In patients at high-risk for developing PA bloodstream infections, the gastrointestinal (GI) tract is the main reservoir for colonization2, but the mechanisms by which PA transitions from an asymptomatic colonizing microbe to an invasive, and often deadly, pathogen are unclear. Previously, we performed in vivo transcription profiling experiments by recovering PA mRNA from bacterial cells residing in the cecums of colonized mice 3 in order to identify changes in bacterial gene expression during alterations to the host&#x2019;s immune status.
As with any transcription profiling experiment, the rate-limiting step is in the isolation of sufficient amounts of high quality mRNA. Given the abundance of enzymes, debris, food residues, and particulate matter in the GI tract, the challenge of RNA isolation is daunting. Here, we present a method for reliable and reproducible isolation of bacterial RNA recovered from the murine GI tract. This method utilizes a well-established murine model of PA GI colonization and neutropenia-induced dissemination4. Once GI colonization with PA is confirmed, mice are euthanized and cecal contents are recovered and flash frozen. RNA is then extracted using a combination of mechanical disruption, boiling, phenol/chloroform extractions, DNase treatment, and affinity chromatography. Quantity and purity are confirmed by spectrophotometry (Nanodrop Technologies) and bioanalyzer (Agilent Technologies) (Fig 1). This method of GI microbial RNA isolation can easily be adapted to other bacteria and fungi as well.

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

A reliable method for the RNA isolation of Pseudomonas aeruginosa recovered from murine cecums is described. The RNA recovered is of sufficient quantity and quality for subsequent qPCR, transcription profiling, and RNA Seq experiments. This technique can be adapted for RNA isolation of other intestinal microbes.

Isolamento de RNA Pseudomonas aeruginosa Colonizar o trato gastrointestinal Murino

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with ...

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable method of analysis by flow cytometry to study nanoparticle interaction with immune cells. Primary immune cells can be easily purified from human or mouse tissues by antibody-mediated magnetic isolation. In the first instance, the different cell populations running in a flow cytometer can be distinguished by the forward-scattered light (FSC), which is proportional to cell size, and the side-scattered light (SSC), related to cell internal complexity. Furthermore, fluorescently labeled antibodies against specific cell surface receptors permit the identification of several subpopulations within the same sample. Often, all these features vary when cells are boosted by external stimuli that change their physiological and morphological state. Here, 50 nm FITC-SiO2 nanoparticles are used as a model to identify the internalization of nanostructured materials in human blood immune cells. The cell fluorescence and side-scattered light increase after incubation with nanoparticles allowed us to define time and concentration dependence of nanoparticle-cell interaction. Moreover, such protocol can be extended to investigate Rhodamine-SiO2 nanoparticle interaction with primary microglia, the central nervous system resident immune cells, isolated from mutant mice that specifically express the Green Fluorescent Protein (GFP) in the monocyte/macrophage lineage. Finally, flow cytometry data related to nanoparticle internalization into the cells have been confirmed by confocal microscopy.

Analysis of nanoparticle interaction with defined subpopulations of immune cells by flow cytometry.

Detecção de nanopartículas fluorescentes Interações com Primário imunitário subpopulações de células por citometria de fluxo

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

Nucleocapsid Annealing-Mediated Electrophoresis (NAME) Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral proteins involved in the regulation of the virus activity recognize several nucleic acids. The nucleocapsid protein NCp7 (NC) is a key protein regulating several processes during virus replication. NC is in fact a chaperone destabilizing the secondary structures of RNA and DNA and facilitating their annealing. The inactivation of NC is a new approach and an interesting target for anti-HIV therapy. The Nucleocapsid Annealing-Mediated Electrophoresis (NAME) assay was developed to identify molecules able to inhibit the melting and annealing of RNA and DNA folded in thermodynamically stable tridimensional conformations, such as hairpin structures of TAR and cTAR elements of HIV, by the nucleocapsid protein of HIV-1. The new assay employs either the recombinant or the synthetic protein, and oligonucleotides without the need of their previous labeling. The analysis of the results is achieved by standard polyacrylamide gel electrophoresis (PAGE) followed by conventional nucleic acid staining. The protocol reported in this work describes how to perform the NAME assay with the full-length protein or its truncated version lacking the basic N-terminal domain, both competent as nucleic acids chaperones, and how to assess the inhibition of NC chaperone activity by a threading intercalator. Moreover, NAME can be performed in two different modes, useful to obtain indications on the putative mechanism of action of the identified NC inhibitors.

Nucleocapsid Annealing-Mediated Electrophoresis NAME Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

This protocol describes NAME, an assay that allows the rapid identification of molecules able to inhibit in vitro the chaperone activities of HIV-1 nucleocapsid protein.

Nucleocápside Annealing-Mediated Eletroforese (NAME) ensaio permite a identificação rápida de HIV-1 Nucleocápside Inibidores

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral ...

Optimization of a Quantitative Micro-neutralization Assay

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. In this study, we present the protocol of an imaging-based MN assay to quantify the true antigenic relationships between viruses. Unlike typical plaque reduction assays that rely on visible plaques, this assay quantitates the entire infected cell population of each well. The protocol matches the virus type or subtype with the selection of cell lines to achieve maximum infectivity, which enhances sample contrast during imaging and image processing. The introduction of quantitative titration defines the amount of input viruses of neutralization and enables the results from different experiments to be comparable. The imaging setup with a flatbed scanner and free downloadable software makes the approach high throughput, cost effective, user friendly, and easy to deploy in most laboratories. Our study demonstrates that the improved MN assay works well with the current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses, without being significantly influenced by amino acid substitutions in the neuraminidase (NA) of A(H3N2) viruses. It is particularly useful for the characterization of viruses that either grow to low HA titer and/or undergo an abortive infection resulting in an inability to form plaques in cultured cells.

This study describes an imaging-based micro-neutralization assay to analyze the antigenic relationships between viruses. The protocol employs a flatbed scanner and has four steps, including titration, titration quantitation, neutralization, and neutralization quantitation. The assay works well with current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses.

Optimização de um Micro-quantitativa Ensaio de neutralização

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. ...

Real-time Quaking-induced Conversion Assay for Detection of CWD Prions in Fecal Material

The RT-QuIC technique is a sensitive in vitro cell-free prion amplification assay based mainly on the seeded misfolding and aggregation of recombinant prion protein (PrP) substrate using prion seeds as a template for the conversion. RT-QuIC is a novel high-throughput technique which is analogous to real-time polymerase chain reaction (PCR). Detection of amyloid fibril growth is based on the dye Thioflavin T, which fluoresces upon specific interaction with &#7526;-sheet rich proteins. Thus, amyloid formation can be detected in real time. We attempted to develop a reliable non-invasive screening test to detect chronic wasting disease (CWD) prions in fecal extract. Here, we have specifically adapted the RT-QuIC technique to reveal PrPSc seeding activity in feces of CWD infected cervids. Initially, the seeding activity of the fecal extracts we prepared was relatively low in RT-QuIC, possibly due to potential assay inhibitors in the fecal material. To improve seeding activity of feces extracts and remove potential assay inhibitors, we homogenized the fecal samples in a buffer containing detergents and protease inhibitors. We also submitted the samples to different methodologies to concentrate PrPSc on the basis of protein precipitation using sodium phosphotungstic acid, and centrifugal force. Finally, the feces extracts were tested by optimized RT-QuIC which included substrate replacement in the protocol to improve the sensitivity of detection. Thus, we established a protocol for sensitive detection of CWD prion seeding activity in feces of pre-clinical and clinical cervids by RT-QuIC, which can be a practical tool for non-invasive CWD diagnosis.

Here, we present a protocol to describe a simple, fast and efficient prion amplification technique, the real-time quaking-induced conversion (RT-QuIC) method.

Em tempo real a tremer induzida por conversão ensaio para detecção de príons CWD em Material Fecal

The RT-QuIC technique is a sensitive in vitro cell-free prion amplification assay based mainly on the seeded misfolding and aggregation of recombinant ...

Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors (PRRs) such as the retinoic acid (RIG-I) like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 (MDA-5) leads to the induction of the innate immune response. The formation and intracellular distribution of dsRNA in positive-sense RNA virus infection has been well characterized by microscopy. Many negative-sense RNA viruses, including some arenaviruses, trigger the innate immune response during infection. However, negative-sense RNA viruses were thought to produce low levels of dsRNA, which hinders the imaging study of PRR recognition of viral dsRNA. Additionally, infection experiments with highly pathogenic arenaviruses must be performed in high containment biosafety level facilities (BSL-4). The interaction between viral RNA and PRRs for highly pathogenic RNA virus is largely unknown due to the additional technical challenges that researchers need to face in the BSL-4 facilities. Recently, a monoclonal antibody (Mab) (clone 9D5) originally used for pan-enterovirus detection has been found to specifically detect dsRNA with a higher sensitivity than the traditional J2 or K1 anti-dsRNA antibodies. Herein, by utilizing the 9D5 antibody, we describe a confocal microscopy protocol that has been used successfully to visualize dsRNA, viral protein and PRR simultaneously in individual cells infected by arenavirus. The protocol is also suitable for imaging studies of dsRNA and PRR distribution in pathogenic arenavirus infected cells in BSL4 facilities.

Double-stranded RNA produced during RNA virus replication can be recognized by pattern recognition receptors to induce an innate immune response. For negative-sense RNA viruses, the interaction between the low-level dsRNA and PRRs remains unclear. We have developed a confocal microscopy method to visualize arenavirus dsRNA and PRR in individual cells.

Imagem latente confocal de RNA Double-Stranded e receptores de reconhecimento padrão na infecção pelo vírus de RNA de sentido negativo

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors ...

An In Vitro Batch-culture Model to Estimate the Effects of Interventional Regimens on Human Fecal Microbiota

The emerging role of the gut microbiome in several human diseases demands a breakthrough of new tools, techniques and technologies. Such improvements are needed to decipher the utilization of microbiome modulators for human health benefits. However, the large-scale screening and optimization of modulators to validate microbiome modulation and predict related health benefits may be practically difficult due to the need for large number of animals and/or human subjects. To this end, in vitro or ex vivo models can facilitate preliminary screening of microbiome modulators. Herein, it is optimized and demonstrated an ex vivo fecal microbiota culture system that can be used for examining the effects of various interventions of gut microbiome modulators including probiotics, prebiotics and other food ingredients, aside from nutraceuticals and drugs, on the diversity and composition of the human gut microbiota. Inulin, one of the most widely studied prebiotic compounds and microbiome modulators, is used as an example here to examine its effect on the healthy fecal microbiota composition and its metabolic activities, such as fecal pH and the fecal levels of organic acids including lactate and short-chain fatty acids (SCFAs). The protocol may be useful for studies aimed at estimating the effects of different interventions of modulators on fecal microbiota profiles and at predicting their health impacts.

This protocol describes an in vitro batch-culture fermentation system of human fecal microbiota, using inulin (a well-known prebiotic and one of the most widely studied microbiota modulators) to demonstrate the use of this system in estimating effects of specific interventions on fecal microbiota composition and metabolic activities.

Um modelo de cultura em lote in vitro para estimar os efeitos dos regimes intervencionistas na microbiota fecal humana

The emerging role of the gut microbiome in several human diseases demands a breakthrough of new tools, techniques and technologies. Such improvements are ...