We received technical assistance from the Biopolymers Facility at Harvard Medical School for bioanalyzer. This work was supported by National Multiple Sclerosis Society research grant RG-1707-28516 (H.L.W. and S.L.).

All methods involving research animals described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of Brigham and Women&#8217;s Hospital, Harvard Medical School.
All methods involving human research subjects described here are in accordance with the guidelines set by the Partners Human Research Committee.
1. Fecal sample collection
<ol>
	<li>Autoclave or prepare a sterile and nuclease-free 2 mL microcentrifuge tube with a screw cap...

<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. 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It is important to use RNase-free technique to prevent RNase contamination during the isolation7. After centrifugation and the formation of a compact interphase, it is key to avoid the interphase, lower phase, and the particle contaminant floating on the top of the aqueous phase when recovering the aqueous phase. Additionally, two washing steps with 500 &#181;L and 250 &#181;L Wash Solution 2/3 are added to eliminate contaminants in the filter membrane for optimized quality. Furthermore, a start s...

Extracellular RNA is getting recognized as a significant factor that mediates many biological processes1. Extracellular RNA in feces was first reported in 2008 as a marker for colon cancer and active ulcerative colitis2, and it was recently revealed as a normal component of the gut lumen and feces and mediates host-microbe communications3,4,5. The purpose of this RNA isolation protocol is to extract high quality extracellular RNA from fecal samples collected from animal and human subjects. The protocol was adapted from a commercial miRNA Isolation Kit. The RNA acquired is utilized for downstream analyses such as RNA sequencing, RT-PCR, and micro-array. The protocol includes several important and useful tips to maximize the quantity and quality of RNA found in the feces of animals and humans. The reason to develop and optimize this method of RNA (including microRNA) isolation is to decrease microbial RNA in the feces, limit the variables in the research studies and analyze the RNA composition in the gut without accounting various confounding factors and sources of contamination. Of note, this RNA isolation minimizes the release of RNA from living cell and living microbes (cellular RNA). It focuses on extracellular RNAs that have been released by gut cells or been acquired via food intake. Principally, this method is not suitable for studies where the microbial transcriptome is investigated.

<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

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 from fecal samples of animal and human subjects. The aim is to isolate total RNA with high purity and quantity for downstream analyses such as RNA sequencing, RT-PCR, and micro-array. The advantages of this optimized protocol in the miRNA isolation are capabilities of isolating highly purified RNA products with additional washing steps described, increased quantity of RNA obtained with an improved method in the resuspension of sample, and important tips of decontamination. One limitation is the inability to process and purify larger sample of more than 200 mg as these sample sizes would cause a difficulty in the clear formation of the interphase. Consequently, the large sample size may contaminate the aqueous phase to be extracted as described in the protocol with organic matters that affect the quality of RNA isolated in the end. However, RNA isolates from a sample of up to 200 mg are sufficient for most of downstream analyses.

Representative RNAs were isolated from 50 mg mouse fecal samples (2 mouse fecal pellets) and 100 mg human stool specimens respectively and eluted in 50 &#181;L nuclease-free water. Spectrophotometer analysis of the concentration suggests a total amount of 49 &#181;g and 16 &#181;g RNA were isolated respectively (Table 1). The RNA purity was high as indicated by an A260/A280 ratio of ~2.0 and an A260/A230 ratio of ~1.8 (Table 1). As reported3, the majority of RNAs ...

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

Fecal micro RNA Isolation

This protocol isolates high quality total RNA from fecal samples of animal and human subjects. A commercial miRNA isolation kit is used with significant adaption to isolate pure RNA with optimized quantity and quality. The RNA isolates are good for most downstream RNA assays such as sequencing, micro-array, and RT-PCR.

Fecal (micro) RNA Isolation

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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4.3K 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.