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

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>
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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 ...

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.

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