Name: fecal (micro) rna isolation
Uploaded: 2020-10-28T15:00:00.000+00:00
Duration: 5 min 35 s
Description: Watch this Scientific Journal Video about Fecal micro RNA Isolation at JoVE.com

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 for each animal subject in an experiment.
	<ol>
		<li>For human subjects, provide an appropriate, nuclease-free, and sterile stool specimen collection device for each subject.</li>
	</ol>
	</li>
	<li>Collect 25-100 mg (approximately 1-4 fecal pellets for mouse fecal samples) of fecal samples from each animal subject in a sterile environment. 
	NOTE: Two or more fecal pellets (~50 mg or heavier) are preferred for obtaining RNA of the highest purity.
	<ol>
		<li>Utilize all necessary Personal Protective Equipment (PPE) and materials, for example: a pair of laboratory gloves, a disinfectant spray and a sterile paper towel, to sterilize the working area, where the animal research subject is placed on, to avoid fecal sample contamination.
		<ol>
			<li>For human subjects, instruct each research subject/collector to collect 100-200 mg of stool samples in an environment as sterile as possible. Use standard sterile operation and avoid stool specimen contamination.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Collect fecal samples from each animal subject directly into a 2 mL microcentrifuge tube with a screw cap without touching any other surfaces to avoid contamination.
	<ol>
		<li>For human subjects, instruct each subject to defecate directly into an applicable collection device provided (e.g., a sterile stool specimen collection kit) to avoid contamination. 
		NOTE: Instruct subject to avoid contamination by toilet surfaces, water, urine, or any other non-sterile surfaces/objects.</li>
	</ol>
	</li>
	<li>Freeze fecal samples collected in 2 mL microcentrifuge tubes immediately at -80 &#176;C or place in a bucket of dry ice for the better quantity and quality of RNA before feces resuspension as described in the steps below.
	<ol>
		<li>For human stool specimens, aliquot each fresh specimen of 200 mg into 2 mL microcentrifuge tubes with screw caps and freeze them at -80 &#176;C before feces resuspension as described below.
		<ol>
			<li>For the storage of stool specimens not in 2 mL microcentrifuge tubes with screw caps, weigh and transfer 100-200 mg each of frozen specimens into separate 2 mL microcentrifuge tubes with screw caps before feces resuspension as described below. 
			NOTE: For human stool specimens, avoid taking more than 200 mg of stool specimens for RNA isolation as it may cause difficulties in the following steps. With an overloaded sample in a 2 mL microcentrifuge tube, the aqueous phase, organic phase and interphase may not be clearly formed and separated. The procedure can be paused here.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Preparations of wash solutions
<ol>
	<li>Add 21 mL of American Chemical Society (ACS) grade 100% ethanol to the Wash Solution 1 provided in the miRNA Isolation Kit&#160;(see Table of Materials)&#160;to reach the final volume of 30 mL, as shown on the bottle. Vortex until everything dissolves in the bottle.</li>
	<li>Add 40 mL of ACS grade 100% ethanol to the Wash Solution 2/3 provided to reach the final volume of 50 mL, as shown on the bottle. Vortex for 5 s or until the final mixture is well blended.</li>
</ol>
3. Preparations of equipment and materials
<ol>
	<li>Spray the working area and equipment, for example: the laboratory bench, the working area in the chemical fume hood and the micro-centrifuge tube racks, with a Ribonuclease (RNase) decontamination solution (e.g., commercially available RNase decontamination solution). To avoid contamination, apply with the RNase decontamination solution on the surfaces wherever and whenever deemed necessary.</li>
	<li>Don a clean laboratory coat, put on a facial mask, and wear appropriate laboratory gloves to protect the RNA in the fecal samples from nucleases present on human skin. Spray gloves with a RNase decontamination solution and change gloves frequently to avoid contamination.</li>
	<li>Prepare a bucket of dry ice for fecal samples stored at -80 &#176;C to prevent thawing before feces resuspension and a bucket of ice for materials, for example: Acid-Phenol: Chloroform, to prolong the shelf life. 
	NOTE: Ensure materials including the media used in the protocol are sterile without contamination of nucleases.</li>
</ol>
4. Feces resuspension
<ol>
	<li>Resuspend 25-100 mg of fecal samples in 600 &#181;L of sterile 1x Dulbecco&#39;s Phosphate-Buffered Saline (DPBS). 
	CAUTION: Fecal samples should be processed immediately when thawed from -80 &#176;C without even partial thawing to minimize the release of RNases and cellular RNA as ice crystals rupture both interior and exterior cellular compartments when cells in sample thaw.
	<ol>
		<li>Add 600 &#181;L of 1x DPBS to the 2 mL microcentrifuge tube with a screw cap containing fecal samples at room temperature (RT).</li>
		<li>Incubate the mixture of fecal samples submerged in 600 &#181;L of 1x DPBS in the 2 mL microcentrifuge tube capped for 30 min at RT.</li>
		<li>Resuspend the mixture by mashing with 1 mL pipette tip and vortex well with the 2 mL microcentrifuge tube capped. To optimize and increase the quantity and quality of RNA, resuspend the mixture with a homogenizer with the setting for one cycle at S4000 (or 4000 rpm) and 45 s.</li>
	</ol>
	</li>
</ol>
5. Organic extraction
CAUTION: Use the hazardous chemical fume hood for the following steps until Step 6 with the use of acid-phenol: chloroform and ACS grade 100% ethanol due to their toxicity and inflammability. Change PPE as needed and follow proper standard precautions when dealing with hazardous material.
<ol>
	<li>Extract RNA with 600 &#181;L of acid-phenol: chloroform (the volume of acid-phenol: chloroform required equals to the initial volume of added 1x DPBS in Step 4.1).
	<ol>
		<li>Add 600 &#956;L of acid-phenol: chloroform to the suspension from Step 4.1. 
		NOTE: Withdraw acid-phenol: chloroform from the lower phase in the bottle as the upper phase is mixed with an aqueous buffer. If the interphase between these two phases is disturbed, then wait and withdraw acid-phenol: chloroform only when the interphase re-establishes itself to avoid contamination.</li>
	</ol>
	</li>
	<li>Vortex the mixture for 60 s to thoroughly mix. Alternatively, to optimize and increase quantity of RNA in the yield, mix by using a homogenizer with the setting for one cycle at S4000 and 45 s.</li>
	<li>Centrifuge for 15 min at 10,000 x g at RT to separate the aqueous and organic phases with a microcentrifuge. After centrifugation, the interphase should be compact. If not, repeat the centrifugation. 
	NOTE: If the interphase could not be as compact as desired possibly due to uneven ratio of the initial volume to the volume of added acid-phenol: chloroform after several repeats of centrifugation, proceed to recover the aqueous phase with a greater care to avoid contamination.</li>
	<li>Recover the aqueous phase and transfer it to a new 2 mL microcentrifuge tube with a hinge cap (not provided by the miRNA isolation kit).
	<ol>
		<li>Remove the aqueous (or upper) phase carefully without disturbing the lower phase and transfer it to a new 2 mL microcentrifuge tube with a hinge cap. Note the volume transferred (e.g., ~500 &#181;L). 
		NOTE: When the interphase is compact and the upper phase is clearly separated, there are possibly a few tiny residual particles floating on the top of the aqueous phase. Pipette carefully to avoid these residues and only recover visibly and clearly separated aqueous phase to ensure a quality RNA yield, even if you could only obtain a small volume of the aqueous phase.</li>
	</ol>
	</li>
</ol>
6. Final RNA isolation
<ol>
	<li>Add 1.25 volumes of RT ACS grade 100% ethanol to the aqueous phase in the 2 mL microcentrifuge tube (e.g., add 625 &#956;L of 100% ethanol if 500 &#956;L of aqueous phase is recovered from Step 5.4.). Vortex 3 s.</li>
	<li>Load the aqueous phase/ethanol mixture through the filter cartridge provided in the miRNA isolation kit.
	<ol>
		<li>For each sample, place the filter cartridge into one of the collection tubes supplied by the kit.
		<ol>
			<li>Pipette and load 600 &#181;L of the aqueous phase/ethanol mixture into the filter cartridge. 
			NOTE: Vortex the mixture briefly to thoroughly mix the ethanol with aqueous phase before pipetting. No more than 700 &#181;L of the aqueous phase/ethanol mixture can be loaded at a time.</li>
		</ol>
		</li>
		<li>Centrifuge at 10,000 x g for 90 s to filter through the mixture. Spinning at a higher speed may damage the filter.</li>
		<li>Discard the filtrate and repeat Steps 6.2.1 to 6.2.2 until all the mixture is filtered through the same filter membrane in successive applications. Keep and reuse the same collection tube for washing steps below.</li>
		<li>Wash the filter with 700 &#181;L of miRNA Wash Solution 1. 
		CAUTION: miRNA Wash Solution 1 contains guanidine thiocyanate that can cause skin irritation and serious eye damage. Wear necessary PPE, for examples: gloves, face shield, protective laboratory coat. Change gloves frequently as necessary.
		<ol>
			<li>Apply 700 &#181;L of miRNA Wash Solution 1, the working solution prepared with the ACS grade 100% ethanol, into the filter cartridge.</li>
			<li>Centrifuge for 60 s to filter the miRNA Wash Solution 1 through the filter cartridge.</li>
			<li>Discard the filtrate from the collection tube and place the same filter cartridge into the same collection tube.</li>
		</ol>
		</li>
		<li>Wash the filter with Wash Solution 2/3 one time each with volumes of 700 &#181;L, 500 &#181;L, and 250 &#181;L consecutively.
		<ol>
			<li>Apply 700 &#181;L of Wash Solution 2/3, the working solution prepared with the ACS grade 100% ethanol, into the filter cartridge.
			<ol>
				<li>Centrifuge at 10,000 x g for 1 min.</li>
				<li>Discard the filtrate from the collection tube and place the same filter cartridge into the same collection tube.</li>
			</ol>
			</li>
			<li>Apply 500 &#181;L of Wash Solution 2/3 into the filter cartridge.
			<ol>
				<li>Centrifuge at 10,000 x g for 1 min.</li>
				<li>Discard the filtrate from the collection tube and place the same filter cartridge into the same collection tube.</li>
			</ol>
			</li>
			<li>Apply 250 &#181;L of Wash Solution 2/3 into the filter cartridge.
			<ol>
				<li>Centrifuge at 10,000 x g for 1 min.</li>
				<li>Discard the filtrate from the collection tube.</li>
			</ol>
			</li>
			<li>Transfer the filter cartridge into a new collection tube and spin the assembly for 5 min to remove residual fluid from the filter.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
7. Elute RNA with 50 &#181;L nuclease-free water
<ol>
	<li>Transfer the filter cartridge into a new collection tube. Pipette 50 &#181;L of nuclease-free water to the center of the filter and cap the collection tube.
	<ol>
		<li>Incubate at RT for 10 min.</li>
		<li>Spin for 5 min at 8,000 x g to recover RNA into the new collection tube.</li>
		<li>Determine the concentration and purity of recovered fecal RNA using a fluorometer. Recovered fecal RNA can be stored at -80 &#730;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. 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The authors declare no relevant or material financial interests that are related to the research method described in this protocol paper.

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 sample material of more than 200 mg is not recommended as it may create difficulty in the clear formation of an interphase. Similarly, a sample material of less than 25 mg is not recommended as it may not be sufficient to extract enough RNA samples for downstream analysis.
The incredible growth in microbiome study has driven the measurements of microbial species, genes to metatranscriptional studies of the microbial profile8. MicroRNAs in the stool have been studied as markers for diseases9,10,11. Since the first report of fecal microRNA mediating host-microbe interactions3, increasing studies start to investigate the contributions of host and diet in the gut ecosystem12,13,14. Noteworthily, due to the richness of microbes in the gut lumen and feces, studies focusing on host arm of the host-microbe interaction demands minimal RNA contamination from microbes. Thus, an RNA extraction protocol that includes steps of cell lysing15 is not ideal for the study of RNAs released from host and diet. As such, we adapted this protocol to eliminate lysis steps for minimizing contaminations of RNA from living bacteria and living host cells in feces.
This protocol works for studies where extracellular RNA in the fecal or gut lumen content is an aim of interest. RNA isolated using this protocol is total RNA, including microRNA as major component. This protocol does not distinguish whether the RNA is in exosome, microvesicles, or in a vesicle-free form.

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 in the feces are microRNA and those microRNAs can exist in the exosome. Consistent with this, 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 (Figure 1A). A further small RNA electrophoresis with the chip-based electrophoresis reveals that a large portion of the RNAs are of microRNA size (Figure 1B).&#160;This is consistent with the observation that the quantification completed by using a small RNA bioanalyzer is comparable to that obtained with previous assays6.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sample ID</td>
			<td>Elution volume (&#181;L)</td>
			<td>RNA concentration (ng/&#181;L)</td>
			<td>A260/A280</td>
			<td>A260/A230</td>
			<td>Yield (ng)</td>
		</tr>
		<tr>
			<td>Mouse</td>
			<td>50</td>
			<td>978.333</td>
			<td>2.036</td>
			<td>1.897</td>
			<td>48916.65</td>
		</tr>
		<tr>
			<td>Human</td>
			<td>50</td>
			<td>330.759</td>
			<td>1.981</td>
			<td>1.849</td>
			<td>16537.95</td>
		</tr>
	</tbody>
</table>
Table 1: Representative nanodrop analysis of RNA isolated with this protocol. Representative RNAs were isolated from 2 mouse fecal pellets or 100 mg human stool specimens, eluted in 50 &#181;L nuclease-free water. RNA concentration, ratio of A260/A280, and ratio of A260/A230 were measured with nanodrop.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61908/61908fig01.jpg" /> 
Figure 1: Representative chip-based electrophoresis analyses of size distribution of fecal RNA isolates.&#160;(A) Representative RNAs isolated from 2 mouse fecal pellets (left panel) and 100 mg human stool specimens (right panel) using the protocol described here were characterized using the chip-based electrophoresis assay.&#160;This assay&#160;suggests that the majority of RNA isolates were small RNA. (B) The isolates were then subjected for the small RNA electrophoresis with the chip-based electrophoresis system to analyze the size distribution of the isolates. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61908/61908fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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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4.5K Views.  Brigham and Women's Hospital and Harvard Medical School. 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.

Video: Fecal micro RNA Isolation

Perturbations of Circulating miRNAs in Irritable Bowel Syndrome Detected Using a Multiplexed High-throughput Gene Expression Platform

The gene expression platform assay allows for robust and highly reproducible quantification of the expression of up to 800 transcripts (mRNA or miRNAs) in a single reaction. The miRNA assay counts transcripts by directly imaging and digitally counting miRNA molecules that are labeled with color-coded fluorescent barcoded probe sets (a reporter probe and a capture probe). Barcodes are hybridized directly to mature miRNAs that have been elongated by ligating a unique oligonucleotide tag (miRtag) to the 3&#39; end. Reverse transcription and amplification of the transcripts are not required. Reporter probes contain a sequence of six color positions populated using a combination of four fluorescent colors. The four colors over six positions are used to construct a gene-specific color barcode sequence. Post-hybridization processing is automated on a robotic prep station. After hybridization, the excess probes are washed away, and the tripartite structures (capture probe-miRNA-reporter probe) are fixed to a streptavidin-coated slide via the biotin-labeled capture probe. Imaging and barcode counting is done using a digital analyzer. The immobilized barcoded miRNAs are visualized and imaged using a microscope and camera, and the unique barcodes are decoded and counted. Data quality control (QC), normalization, and analysis are facilitated by a custom-designed data processing and analysis software that accompanies the assay software. The assay demonstrates high linearity over a broad range of expression, as well as high sensitivity. Sample and assay preparation does not involve enzymatic reactions, reverse transcription, or amplification; has few steps; and is largely automated, reducing investigator effects and resulting in high consistency and technical reproducibility. Here, we describe the application of this technology to identifying circulating miRNA perturbations in irritable bowel syndrome.

We describe the use of a multiplexed high-throughput gene expression platform that quantitates gene expression by barcoding and counting molecules in biological substrates without the need for amplification. We used the platform to quantitate microRNA (miRNA) expression in whole blood in subjects with and without irritable bowel syndrome.

The gene expression platform assay allows for robust and highly reproducible quantification of the expression of up to 800 transcripts (mRNA or miRNAs) in ...

Genetics

Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens

&#8211;Archived, clinically classified formalin-fixed paraffin-embedded (FFPE) tissues can provide nucleic acids for retrospective molecular studies of cancer development. By using non-invasive or pre-malignant lesions from patients who later develop invasive disease, gene expression analyses may help identify early molecular alterations that predispose to cancer risk. It has been well described that nucleic acids recovered from FFPE tissues have undergone severe physical damage and chemical modifications, which make their analysis difficult and generally requires adapted assays. MicroRNAs (miRNAs), however, which represent a small class of RNA molecules spanning only up to ~18&#8211;24 nucleotides, have been shown to withstand long-term storage and have been successfully analyzed in FFPE samples. Here we present a 3&#39; barcoded complementary DNA (cDNA) library preparation protocol specifically optimized for the analysis of small RNAs extracted from archived tissues, which was recently demonstrated to be robust and highly reproducible when using archived clinical specimens stored for up to 35 years. This library preparation is well adapted to the multiplex analysis of compromised/degraded material where RNA samples (up to 18) are ligated with individual 3&#39; barcoded adapters and then pooled together for subsequent enzymatic and biochemical preparations prior to analysis. All purifications are performed by polyacrylamide gel electrophoresis (PAGE), which allows size-specific selections and enrichments of barcoded small RNA species. This cDNA library preparation is well adapted to minute RNA inputs, as a pilot polymerase chain reaction (PCR) allows determination of a specific amplification cycle to produce optimal amounts of material for next-generation sequencing (NGS). This approach was optimized for the use of degraded FFPE RNA from specimens archived for up to 35 years and provides highly reproducible NGS data.

Formalin-fixed paraffin-embedded specimens represent a valuable source of molecular biomarkers of human diseases. Here we present a laboratory-based cDNA library preparation protocol, initially designed with fresh frozen RNA, and optimized for the analysis of archived microRNAs from tissues stored up to 35 years.

&#8211;Archived, clinically classified formalin-fixed paraffin-embedded (FFPE) tissues can provide nucleic acids for retrospective molecular studies of ...

Isolating, Sequencing and Analyzing Extracellular MicroRNAs from Human Mesenchymal Stem Cells

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, milk and urine. One subset of these RNAs are the posttranscriptional regulators &#8211; microRNAs (miRNAs). To delineate the miRNAs produced by specific cell types, in vitro culture systems can be used to harvest and profile exRNAs derived from one subset of cells. The secreted factors of mesenchymal stem cells are implicated in alleviating numerous diseases and is used as the in vitro model system here. This paper describes the process of collection, purification of small RNA and library generation to sequence extracellular miRNAs. ExRNAs from culture media differ from cellular RNA by being low RNA input samples, which calls for optimized procedures. This protocol provides a comprehensive guide to small exRNA sequencing from culture media, showing quality control checkpoints at each step during exRNA purification and sequencing.

This protocol demonstrates how to purify extracellular microRNAs from cell culture media for small RNA library construction and next generation sequencing. Various quality control checkpoints are described to allow readers to understand what to expect when working with low input samples like exRNAs.

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, ...

A Complete Pipeline for Isolating and Sequencing MicroRNAs, and Analyzing Them Using Open Source Tools

Half of all human transcripts are thought to be regulated by microRNAs. Therefore, quantifying microRNA expression can reveal underlying mechanisms in disease states and provide therapeutic targets and biomarkers. Here, we detail how to accurately quantify microRNAs. Briefly, this method describes isolating microRNAs, ligating them to adaptors suitable for high-throughput sequencing, amplifying the final products, and preparing a sample library. Then, we explain how to align the obtained sequencing reads to microRNA hairpins, and quantify, normalize, and calculate their differential expression. Versatile and robust, this combined experimental workflow and bioinformatic analysis enables users to begin with tissue extraction and finish with microRNA quantification.

Here, we describe a step-by-step strategy for isolating small RNAs, enriching for microRNAs, and preparing samples for high-throughput sequencing. We then describe how to process sequence reads and align them to microRNAs, using open source tools.

Half of all human transcripts are thought to be regulated by microRNAs. Therefore, quantifying microRNA expression can reveal underlying mechanisms in ...

Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their translational repression. Changes in miRNA expression have been shown to be involved in the development of all major complex diseases. Furthermore, recent findings showed that miRNAs can be secreted to the extracellular environment and enter the bloodstream and other body fluids where they can circulate with high stability. The function of such circulating miRNAs remains largely elusive, but systematic high throughput approaches, such as miRNA profiling arrays, have lead to the identification of miRNA signatures in several pathological conditions, including neurodegenerative disorders and several types of cancers. In this context, the identification of miRNA expression profile in the cerebrospinal fluid, as reported in our recent study, makes miRNAs attractive candidates for biomarker analysis.
There are several tools available for profiling microRNAs, such as microarrays, quantitative real-time PCR (qPCR), and deep sequencing. Here, we describe a sensitive method to profile microRNAs in cerebrospinal fluids by quantitative real-time PCR. We used the Exiqon microRNA ready-to-use PCR human panels I and II V2.R, which allows detection of 742 unique human microRNAs. We performed the arrays in triplicate runs and we processed and analyzed data using the GenEx Professional 5 software.
Using this protocol, we have successfully profiled microRNAs in various types of cell lines and primary cells, CSF, plasma, and formalin-fixed paraffin-embedded tissues.

We describe a protocol of real time PCR to profile microRNAs in the cerebrospinal fluid (CSF). With the exception of RNA extraction protocols, the procedure can be extended to RNA extracted from other body fluids, cultured cells, or tissue specimens.

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their ...

Medicine

Circulating MicroRNA Quantification Using DNA-binding Dye Chemistry and Droplet Digital PCR

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been linked to a disease state and has the potential to be used as disease biomarker. A sensitive and accurate method for circulating microRNA quantification using a dye-based chemistry and droplet digital PCR technology has been recently developed. Specifically, using Locked Nucleic Acid (LNA)-based miRNA-specific primers with a green fluorescent DNA-binding dye in a compatible droplet digital PCR system it is possible to obtain the absolute quantification of specific miRNAs. Here, we describe how performing this technique to assess miRNA amount in biological fluids, such as plasma and serum, is both feasible and effective.

A sensitive and accurate method for cell-free microRNAs quantification using a dye-based chemistry and droplet digital PCR technology is described.

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been ...

Bioengineering

Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic uses.
Exosomes have recently been shown to contain microRNAs (miRs). MiRs themselves have arisen as valuable biomarkers for diagnostic purposes. As specimen collection in clinics and hospitals is quite variable, miRNA isolation from whole bile varies substantially. To achieve robust, accurate and reproducible miRNA profiles from collected bile samples in a simple manner required the development of a high-quality protocol to isolate and characterize exosomes from bile. The method requires several centrifugations and a filtration step with a final ultracentrifugation step to pellet the isolated exosomes. Electron microscopy, Western blots, flow cytometry and multi-parameter nanoparticle optical analysis, where available, are crucial characterization steps to validate the quality of the exosomes. For the isolation of miRNA from these exosomes, spiking the lysate with a non-specific, synthetic miRNA from a species like Caenorhabditis elegans, i.e., Cel-miR-39, is important for normalization of RNA extraction efficiency. The isolation of exosome from bile fluid following this method allows the successful miRNA profiling from bile samples stored for several years at -80 &#176;C.

Bile fluid is a valuable source of extracellular vesicles/exosomes that contain potentially important biomarkers. This protocol represents a robust method to isolate exosomes from human bile for further analyses including miRNA profiling.

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic ...

Purification and microRNA Profiling of Exosomes Derived from Blood and Culture Media

Stable miRNAs are present in all body fluids and some circulating miRNAs are protected from degradation by sequestration in small vesicles called exosomes. Exosomes can fuse with the plasma membrane resulting in the transfer of RNA and proteins to the target cell. Their biological functions include immune response, antigen presentation, and intracellular communication. Delivery of miRNAs that can regulate gene expression in the recipient cells via blood has opened novel avenues for target intervention. In addition to offering a strategy for delivery of drugs or RNA therapeutic agents, exosomal contents can serve as biomarkers that can aid in diagnosis, determining treatment options and prognosis.	Here we will describe the procedure for quantitatively analyzing miRNAs and messenger RNAs (mRNA) from exosomes secreted in blood and cell culture media. Purified exosomes will be characterized using western blot analysis for exosomal markers and PCR for mRNAs of interest. Transmission electron microscopy (TEM) and immunogold labeling will be used to validate exosomal morphology and integrity. Total RNA will be purified from these exosomes to ensure that we can study both mRNA and miRNA from the same sample. After validating RNA integrity by Bioanalyzer, we will perform a medium throughput quantitative real time PCR (qPCR) to identify the exosomal miRNA using Taqman Low Density Array (TLDA) cards and gene expression studies for transcripts of interest.	
 These protocols can be used to quantify changes in exosomal miRNAs in patients, rodent models and cell culture media before and after pharmacological intervention. Exosomal contents vary due to the source of origin and the physiological conditions of cells that secrete exosomes. These variations can provide insight on how cells and systems cope with stress or physiological perturbations. Our representative data show variations in miRNAs present in exosomes purified from mouse blood, human blood and human cell culture media.	
 Here we will describe the procedure for quantitatively analyzing miRNAs and messenger RNAs (mRNA) from exosomes secreted in blood and cell culture media. Purified exosomes will be characterized using western blot analysis for exosomal markers and PCR for mRNAs of interest. Transmission electron microscopy (TEM) and immunogold labeling will be used to validate exosomal morphology and integrity. Total RNA will be purified from these exosomes to ensure that we can study both mRNA and miRNA from the same sample. After validating RNA integrity by Bioanalyzer, we will perform a medium throughput quantitative real time PCR (qPCR) to identify the exosomal miRNA using Taqman Low Density Array (TLDA) cards and gene expression studies for transcripts of interest.
 These protocols can be used to quantify changes in exosomal miRNAs in patients, rodent models and cell culture media before and after pharmacological intervention. Exosomal contents vary due to the source of origin and the physiological conditions of cells that secrete exosomes. These variations can provide insight on how cells and systems cope with stress or physiological perturbations. Our representative data show variations in miRNAs present in exosomes purified from mouse blood, human blood and human cell culture media

The presence of stable microRNAs (miRNAs) in exosomes has generated immense interest as a novel mode of intercellular communication, for their potential utility as biomarkers and as a route for therapeutic intervention. Here we demonstrate exosome purification from blood and culture media followed by quantitative PCR to identify miRNAs being transported.

Stable miRNAs are present in all body fluids and some circulating miRNAs  are protected from degradation by sequestration in small vesicles called  ...

Biology

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the immune system. Perturbation of the healthy gut microbiome has been suggested to play a role in the development of inflammatory diseases, including multiple sclerosis (MS). Environmental and genetic factors can influence the composition of the microbiome; therefore, identification of microbial communities linked with a disease phenotype has become the first step towards defining the microbiome&#8217;s role in health and disease. Use of 16S rRNA metagenomic sequencing for profiling bacterial community has helped in advancing microbiome research. Despite its wide use, there is no uniform protocol for 16S rRNA-based taxonomic profiling analysis. Another limitation is the low resolution of taxonomic assignment due to technical difficulties such as smaller sequencing reads, as well as use of only forward (R1) reads in the final analysis due to low quality of reverse (R2) reads. There is need for a simplified method with high resolution to characterize bacterial diversity in a given biospecimen. Advancements in sequencing technology with the ability to sequence longer reads at high resolution have helped to overcome some of these challenges. Present sequencing technology combined with a publicly available metagenomic analysis pipeline such as R-based Divisive Amplicon Denoising Algorithm-2 (DADA2) has helped advance microbial profiling at high resolution, as DADA2 can assign sequence at the genus and species levels. Described here is a guide for performing bacterial profiling using two-step amplification of the V3-V4 region of the 16S rRNA gene, followed by analysis using freely available analysis tools (i.e., DADA2, Phyloseq, and METAGENassist). It is believed that this simple and complete workflow will serve as an excellent tool for researchers interested in performing microbiome profiling studies.

Described here is a simplified standard operating procedure for microbiome profiling using 16S rRNA metagenomic sequencing and analysis using freely available tools. This protocol will help researchers who are new to the microbiome field as well as those requiring updates on methods to achieve bacterial profiling at a higher resolution.

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the ...

Isolation of microRNAs from Tick Ex Vivo Salivary Gland Cultures and Extracellular Vesicles

Ticks are important ectoparasites that can vector multiple pathogens. The salivary glands of ticks are essential for feeding as their saliva contains many effectors with pharmaceutical properties that can diminish host immune responses and enhance pathogen transmission. One group of such effectors are microRNAs (miRNAs). miRNAs are short non-coding sequences that regulate host gene expression at the tick-host interface and within the organs of the tick. These small RNAs are transported in the tick saliva via extracellular vesicles (EVs), which serve inter-and intracellular communication. Vesicles containing miRNAs have been identified in the saliva of ticks. However, little is known about the roles and profiles of the miRNAs in tick salivary vesicles and glands. Furthermore, the study of vesicles and miRNAs in tick saliva requires tedious procedures to collect tick saliva. This protocol aims to develop and validate a method for isolating miRNAs from purified extracellular vesicles produced by ex vivo organ cultures. The materials and methodology needed to extract miRNAs from extracellular vesicles and tick salivary glands are described herein.

Isolation of microRNAs from Tick Ex Vivo Salivary Gland Cultures and Extracellular Vesicles

The present protocol describes the isolation of microRNAs from tick salivary glands and purified extracellular vesicles. This is a universal procedure that combines commonly used reagents and supplies. The method also allows the use of a small number of ticks, resulting in quality microRNAs that can be readily sequenced.

Ticks are important ectoparasites that can vector multiple pathogens. The salivary glands of ticks are essential for feeding as their saliva contains many ...

Fecal (micro) RNA Isolation

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Chapters in this video

Perturbations of Circulating miRNAs in Irritable Bowel Syndrome Detected Using a Multiplexed High-throughput Gene Expression Platform

Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens

Isolating, Sequencing and Analyzing Extracellular MicroRNAs from Human Mesenchymal Stem Cells

A Complete Pipeline for Isolating and Sequencing MicroRNAs, and Analyzing Them Using Open Source Tools

Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR

Circulating MicroRNA Quantification Using DNA-binding Dye Chemistry and Droplet Digital PCR

Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

Purification and microRNA Profiling of Exosomes Derived from Blood and Culture Media

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

Isolation of microRNAs from Tick Ex Vivo Salivary Gland Cultures and Extracellular Vesicles