Name: fecal (micro) rna isolation
Uploaded: 2020-10-28T15:00:00
Description: Watch this Scientific Journal Video about Fecal micro RNA Isolation at JoVE.com

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 l...

<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>

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)...

Watch this Scientific Journal Video about Fecal micro RNA Isolation at JoVE.com

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 ...

We present a protocol for isolating RNA from fecal samples to study microRNAs. This protocol can be used to isolate RNA from feces with high quality and quantity. It minimizes RNAs from living microbes.It is important to emphasize that and the binding buffer is not used in this protocol. This protocol is simple and straightforward. Begin by resuspending 25 to 100 milligrams of fecal samples in 600 microliters of sterile 1X DPBS in a two-milliliter microcentrifuge tube.Incubate the tube containing the sample at room temperature for 30 minutes. Then mash the mixture with a one milliliter pipette tip and vortex well. Resuspend the mixture in a homogenizer with a setting for one cycle at 4, 000 rotations per minute for 45 seconds to optimize and increase the quantity and quality of RNA.Add 600 microliters of acid phenol chloroform solution to the sample and vortex the mixture for 60 seconds. Alternatively, to optimize an increased quantity of RNA in the yield, mix it with a homogenizer. Centrifuge the sample for 15 minutes at 10, 000 x g at room temperature to separate the aqueous and organic phase.Repeat the centrifugation until the interface is compact. Transfer the upper aqueous phase carefully without disturbing the lower phase to a new two milliliter microcentrifuge tube with a hinge cap. Add ACS grade 100%ethanol at 1.25 x the volume of the acquired aqueous phase and vortex for three seconds.Place the filter cartridge into the collection tubes. Load 600 microliters of each of the samples in the filter cartridge provided in the microRNA isolation kit. Centrifuge at 10, 000 x g for 90 seconds to filter the mixture through the cartridge.Then discard the filtrate. Repeat this until the entire volume of the mixture is filtered through the same filter membrane in successive applications. After all the sample is filtered.add 700 microliters of microRNA wash solution 1 into the same filter cartridge. Centrifuge for 60 seconds to filter the wash solution through the filter cartridge. Discard the filtrate and place the filter cartridge on the same collection tube.Wash the filter cartridge with 700 microliters of wash solution 2/3 prepared in ACS grade 100%ethanol and centrifuge at 10, 000 x g for a minute. Discard the obtained filtrate and place the filter cartridge into the same tube. Wash the filter cartridge with 500 microliters of wash solution 2/3 and centrifuge at 10, 000 x g for a minute.Discard the obtained filtrate and place the filter cartridge into the same collection tube. Wash the filter cartridge with 250 microliters of wash solution 2/3 prepared in ACS grade 100%ethanol and centrifuge at 10, 000 x g for a minute. Discard the obtained filtrate and place the filter cartridge into the same collection tube.Finally, transfer the filter cartridge into a new collection tube and spin the assembly for five minutes to remove the residual fluid. Transfer the filter cartridge into a new collection tube then add 50 microliters of nuclease-free water to the center of the filter and cap the tube. Incubate the tube at room temperature for 10 minutes.Then spin the tube for five minutes at 8, 000 x g to recover the RNA into a new collection tube. A chip-based electrophoresis assay of RNA suggests that representative RNA isolates from mouse and human feces are low in or lack of 18S and 28S rRNA compositions and the size of RNA isolates falls in the small RNA region. A further small RNA electrophoresis with the chip-based electrophoresis revealed that a large portion of the RNAs are of microRNA size.The purity of the extracted RNA was high as indicated by the absorbance ratio of 2.0 for 260 and 280-nanometer wavelengths and 1.8 ratio value for absorbance at 260 and 230 nanometers. To ensure success of the organic extraction, make sure that the faces are obvious and only transfer the upper aqueous phase without disturbing the lower phase, even at the cost of a reduced volume of our aqueous phase.

Research

JoVE Journal

Immunology and Infection

Fecal (micro) RNA Isolation

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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 ...

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

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 ...

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

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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

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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

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

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

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 ...

Culture Methods to Determine the Limit of Detection and Survival in Transport Media of Campylobacter Jejuni in Human Fecal Specimens

Culture Methods to Determine the Limit of Detection and Survival in Transport Media of Campylobacter Jejuni in Human Fecal Specimens

Métodos de Cultura para determinar o limite de detecção e sobrevivência em mídia de transporte de Campylobacter Jejuni em espécimes fecais humanos

A culture from human stool for diagnosis of Campylobacter-based intestinal illness takes several days, a wait that taxes the fortitude of the physician ...

Supervised Machine Learning for Semi-Quantification of Extracellular DNA in Glomerulonephritis

Aprendizado de Máquina Supervisionado para Semi-Quantificação de DNA Extracelular em Glomerulonephritis

Glomerular cell death is a pathological feature of myeloperoxidase anti neutrophil cytoplasmic antibody associated vasculitis (MPO-AAV). Extracellular ...

Therapeutic Evaluation of Fecal Microbiota Transplantation in an Interleukin 10-Deficient Mouse Model

Avaliação Terapêutica do Transplante de Microbiota Fecal em um Modelo de Camundongo deficiente interleucina

With the development of microecology in recent years, the relationship between intestinal bacteria and inflammatory bowel disease (IBD) has attracted ...