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

Vi modtog teknisk assistance fra Biopolymers Facility på Harvard Medical School til bioanalysator. Dette arbejde blev støttet af National Multiple Sclerosis Society forskningsbevilling RG-1707-28516 (H.L.W. og S.L.).

Alle metoder, der involverer forsøgsdyr, der er beskrevet her, er blevet godkendt af Institutional Animal Care and Use Committee (IACUC) på Brigham and Women's Hospital, Harvard Medical School.
Alle metoder, der involverer menneskelige forskningsemner, der er beskrevet her, er i overensstemmelse med de retningslinjer, der er fastsat af Partners Human Research Committee.
1. Indsamling af fækal prøve
<ol><li>Autoklave eller tilbered et sterilt og nukleasefrit 2 ...

<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>

Det er vigtigt at bruge RNase-fri teknik for at forhindre RNase-forurening under isoleringen7. Efter centrifugering og dannelsen af en kompakt interfase er det nøglen til at undgå, at interfasen, den nedre fase og partikelforureningen flyder på toppen af den vandige fase, når man genvinder den vandige fase. Derudover tilføjes to vasketrin med 500 μL og 250 μL vaskeopløsning 2/3 for at eliminere forurenende stoffer i filtermembranen for optimeret kvalitet. Desuden anbefales et startprøvema...

Ekstracellulært RNA bliver anerkendt som en væsentlig faktor, der formidler mange biologiske processer1. Ekstracellulært RNA i afføring blev først rapporteret i 2008 som en markør for tyktarmskræft og aktiv ulcerøs colitis2, og det blev for nylig afsløret som en normal komponent i tarmens lumen og afføring og medierer værtsmikrobekommunikation 3,4,5. Formålet med denne RNA-isolationsprotokol er at ekstracellulære RNA af høj kvalitet fra fækale prøver indsamlet fra dyr og mennesker. Protokollen blev tilpasset fra et kommercielt miRNA-isolationssæt. Det erhvervede RNA bruges til downstream-analyser såsom RNA-sekventering, RT-PCR og mikroarray. Protokollen indeholder flere vigtige og nyttige tips til at maksimere mængden og kvaliteten af RNA, der findes i afføring fra dyr og mennesker. Årsagen til at udvikle og optimere denne metode til RNA (herunder microRNA) isolering er at reducere mikrobielt RNA i afføringen, begrænse variablerne i forskningsundersøgelserne og analysere RNA-sammensætningen i tarmen uden at redegøre for forskellige forvirrende faktorer og forureningskilder. Bemærk, at denne RNA-isolering minimerer frigivelsen af RNA fra levende celler og levende mikrober (cellulært RNA). Det fokuserer på ekstracellulære RNA'er, der er blevet frigivet af tarmceller eller er blevet erhvervet via fødeindtagelse. Principielt er denne metode ikke egnet til undersøgelser, hvor det mikrobielle transkriptom undersøges.

<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

Det bliver klart, at RNA findes i tarmens lumen og afføring hos dyr og mennesker. Protokollen beskrevet nedenfor isolerer total RNA inklusive mikroRNA'er fra fækale prøver af dyr og mennesker. Målet er at isolere total RNA med høj renhed og mængde til downstream-analyser såsom RNA-sekventering, RT-PCR og mikroarray. Fordelene ved denne optimerede protokol i miRNA-isoleringen er muligheder for at isolere højt oprensede RNA-produkter med yderligere vasketrin beskrevet, øget mængde RNA opnået med en forbedret metode til resuspension af prøve og vigtige tip til dekontaminering. En begrænsning er manglende evne til at behandle og rense større prøve på mere end 200 mg, da disse prøvestørrelser ville forårsage vanskeligheder med den klare dannelse af interfasen. Følgelig kan den store prøvestørrelse forurene den vandige fase, der skal ekstraheres som beskrevet i protokollen med organiske stoffer, der påvirker kvaliteten af RNA isoleret i sidste ende. RNA-isolater fra en prøve på op til 200 mg er imidlertid tilstrækkelige til de fleste downstream-analyser.

Repræsentative RNA'er blev isoleret fra henholdsvis 50 mg musefækale prøver (2 musefækale pellets) og 100 mg humane afføringsprøver og elueret i 50 μL nukleasefrit vand. Spektrofotometeranalyse af koncentrationen antyder, at en samlet mængde på henholdsvis 49 μg og 16 μg RNA blev isoleret (tabel 1). RNA-renheden var høj som angivet ved et A260/A280-forhold på ~2,0 og et A260/A230-forhold på ~1,8 (tabel 1). Som rapporteret3 er størstedelen af RNA'ern...

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

Fecal micro RNA Isolation

Denne protokol isolerer total RNA af høj kvalitet fra fækale prøver af dyr og mennesker. Et kommercielt miRNA-isolationssæt bruges med betydelig tilpasning til at isolere rent RNA med optimeret mængde og kvalitet. RNA-isolaterne er gode til de fleste downstream RNA-assays såsom sekventering, mikroarray og RT-PCR.

Fækal (mikro) RNA-isolering

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 ...

Research

JoVE Journal

Immunology and Infection

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 ...

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 ...

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

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 ...

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

Påvisning af fluorescerende Nanopartikel Interaktioner med Primary Immune cellesubpopulationer ved flowcytometri

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

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

Nucleocapsid Annealing-medieret Electrophoresis (NAME) Indhold Tillader hurtig identifikation af HIV-1 nucleocapsid inhibitorer

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

Optimering af en kvantitativ Micro-neutraliseringsanalyse

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

Real-time skælven-induceret konvertering Assay til påvisning af CWD prioner i Fecal materiale

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

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

Kultur metoder til at bestemme grænsen for påvisning og overlevelse i Transport Media af Campylobacter Jejuni i Human Fecal Prøver

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

Overvåget maskinlæring til semi-kvantificering af ekstracellulær DNA i glomerulonefritis

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

Terapeutisk evaluering af fækal mikrobiotatransplantation i en Interleukin 10-mangelfuld musemodel

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