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>
	<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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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.

Research

JoVE Journal

Immunology and Infection

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 of human diseases1 and the most important vectors affecting cattle industry worldwide2. Ticks are classified in the subclass Acari, order Parasitiformes, suborder Ixodida and are distributed worldwide from Arctic to tropical regions3. Despite efforts to control tick infestations, these ectoparasites remain a serious problem for human and animal health4,5.
RNA interference (RNAi)6 is a nucleic acid-based reverse genetic approach that involves disruption of gene expression in order to determine gene function or its effect on a metabolic pathway. Small interfering RNAs (siRNAs) are the effector molecules of the RNAi pathway that is initiated by double-stranded RNA (dsRNA) and results in a potent sequence-specific degradation of cytoplasmic mRNAs containing the same sequence as the dsRNA trigger7-9. Post-transcriptional gene silencing mechanisms initiated by dsRNA have been discovered in all eukaryotes studied thus far, and RNAi has been rapidly developed in a variety of organisms as a tool for functional genomics studies and other applications10.
RNAi has become the most widely used gene-silencing technique in ticks and other organisms where alternative approaches for genetic manipulation are not available or are unreliable5,11. The genetic characterization of ticks has been limited until the recent application of RNAi12,13. In the short time that RNAi has been available, it has proved to be a valuable tool for studying tick gene function, the characterization of the tick-pathogen interface and the screening and characterization of tick protective antigens14. Herein, a method for RNAi through injection of dsRNA into unfed ticks is described. It is likely that the knowledge gained from this experimental approach will contribute markedly to the understanding of basic biological systems and the development of vaccines to control tick infestations and prevent transmission of tick-borne pathogens15-19.

A method for RNA interference (RNAi) by injection of dsRNA into unfed ticks is described. RNAi is the most widely used gene-silencing technique in ticks where the use of other methods of genetic manipulation has been limited.

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 generation of extreme population diversity that facilitates virus adaptation and evolution. Increasing evidence shows that the intrinsic error rates, and the resulting mutation frequencies, of RNA viruses can be modulated by subtle amino acid changes to the viral polymerase. Although biochemical assays exist for some viral RNA polymerases that permit quantitative measure of incorporation fidelity, here we describe a simple method of measuring mutation frequencies of RNA viruses that has proven to be as accurate as biochemical approaches in identifying fidelity altering mutations. The approach uses conventional virological and sequencing techniques that can be performed in most biology laboratories. Based on our experience with a number of different viruses, we have identified the key steps that must be optimized to increase the likelihood of isolating fidelity variants and generating data of statistical significance. The isolation and characterization of fidelity altering mutations can provide new insights into polymerase structure and function1-3. Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution4-7.

The present article describes the steps required to isolate and characterize RNA polymerase fidelity variants of RNA viruses and how to use mutation frequency data to confirm fidelity changes in tissue culture.

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

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with leukemia, severe burn wounds, or organ transplants1. In patients at high-risk for developing PA bloodstream infections, the gastrointestinal (GI) tract is the main reservoir for colonization2, but the mechanisms by which PA transitions from an asymptomatic colonizing microbe to an invasive, and often deadly, pathogen are unclear. Previously, we performed in vivo transcription profiling experiments by recovering PA mRNA from bacterial cells residing in the cecums of colonized mice 3 in order to identify changes in bacterial gene expression during alterations to the host&#x2019;s immune status.
As with any transcription profiling experiment, the rate-limiting step is in the isolation of sufficient amounts of high quality mRNA. Given the abundance of enzymes, debris, food residues, and particulate matter in the GI tract, the challenge of RNA isolation is daunting. Here, we present a method for reliable and reproducible isolation of bacterial RNA recovered from the murine GI tract. This method utilizes a well-established murine model of PA GI colonization and neutropenia-induced dissemination4. Once GI colonization with PA is confirmed, mice are euthanized and cecal contents are recovered and flash frozen. RNA is then extracted using a combination of mechanical disruption, boiling, phenol/chloroform extractions, DNase treatment, and affinity chromatography. Quantity and purity are confirmed by spectrophotometry (Nanodrop Technologies) and bioanalyzer (Agilent Technologies) (Fig 1). This method of GI microbial RNA isolation can easily be adapted to other bacteria and fungi as well.

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

A reliable method for the RNA isolation of Pseudomonas aeruginosa recovered from murine cecums is described. The RNA recovered is of sufficient quantity and quality for subsequent qPCR, transcription profiling, and RNA Seq experiments. This technique can be adapted for RNA isolation of other intestinal microbes.

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

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 proteins involved in the regulation of the virus activity recognize several nucleic acids. The nucleocapsid protein NCp7 (NC) is a key protein regulating several processes during virus replication. NC is in fact a chaperone destabilizing the secondary structures of RNA and DNA and facilitating their annealing. The inactivation of NC is a new approach and an interesting target for anti-HIV therapy. The Nucleocapsid Annealing-Mediated Electrophoresis (NAME) assay was developed to identify molecules able to inhibit the melting and annealing of RNA and DNA folded in thermodynamically stable tridimensional conformations, such as hairpin structures of TAR and cTAR elements of HIV, by the nucleocapsid protein of HIV-1. The new assay employs either the recombinant or the synthetic protein, and oligonucleotides without the need of their previous labeling. The analysis of the results is achieved by standard polyacrylamide gel electrophoresis (PAGE) followed by conventional nucleic acid staining. The protocol reported in this work describes how to perform the NAME assay with the full-length protein or its truncated version lacking the basic N-terminal domain, both competent as nucleic acids chaperones, and how to assess the inhibition of NC chaperone activity by a threading intercalator. Moreover, NAME can be performed in two different modes, useful to obtain indications on the putative mechanism of action of the identified NC inhibitors.

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

This protocol describes NAME, an assay that allows the rapid identification of molecules able to inhibit in vitro the chaperone activities of HIV-1 nucleocapsid protein.

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

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. In this study, we present the protocol of an imaging-based MN assay to quantify the true antigenic relationships between viruses. Unlike typical plaque reduction assays that rely on visible plaques, this assay quantitates the entire infected cell population of each well. The protocol matches the virus type or subtype with the selection of cell lines to achieve maximum infectivity, which enhances sample contrast during imaging and image processing. The introduction of quantitative titration defines the amount of input viruses of neutralization and enables the results from different experiments to be comparable. The imaging setup with a flatbed scanner and free downloadable software makes the approach high throughput, cost effective, user friendly, and easy to deploy in most laboratories. Our study demonstrates that the improved MN assay works well with the current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses, without being significantly influenced by amino acid substitutions in the neuraminidase (NA) of A(H3N2) viruses. It is particularly useful for the characterization of viruses that either grow to low HA titer and/or undergo an abortive infection resulting in an inability to form plaques in cultured cells.

This study describes an imaging-based micro-neutralization assay to analyze the antigenic relationships between viruses. The protocol employs a flatbed scanner and has four steps, including titration, titration quantitation, neutralization, and neutralization quantitation. The assay works well with current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses.

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

The RT-QuIC technique is a sensitive in vitro cell-free prion amplification assay based mainly on the seeded misfolding and aggregation of recombinant prion protein (PrP) substrate using prion seeds as a template for the conversion. RT-QuIC is a novel high-throughput technique which is analogous to real-time polymerase chain reaction (PCR). Detection of amyloid fibril growth is based on the dye Thioflavin T, which fluoresces upon specific interaction with &#7526;-sheet rich proteins. Thus, amyloid formation can be detected in real time. We attempted to develop a reliable non-invasive screening test to detect chronic wasting disease (CWD) prions in fecal extract. Here, we have specifically adapted the RT-QuIC technique to reveal PrPSc seeding activity in feces of CWD infected cervids. Initially, the seeding activity of the fecal extracts we prepared was relatively low in RT-QuIC, possibly due to potential assay inhibitors in the fecal material. To improve seeding activity of feces extracts and remove potential assay inhibitors, we homogenized the fecal samples in a buffer containing detergents and protease inhibitors. We also submitted the samples to different methodologies to concentrate PrPSc on the basis of protein precipitation using sodium phosphotungstic acid, and centrifugal force. Finally, the feces extracts were tested by optimized RT-QuIC which included substrate replacement in the protocol to improve the sensitivity of detection. Thus, we established a protocol for sensitive detection of CWD prion seeding activity in feces of pre-clinical and clinical cervids by RT-QuIC, which can be a practical tool for non-invasive CWD diagnosis.

Here, we present a protocol to describe a simple, fast and efficient prion amplification technique, the real-time quaking-induced conversion (RT-QuIC) method.

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

The emerging role of the gut microbiome in several human diseases demands a breakthrough of new tools, techniques and technologies. Such improvements are needed to decipher the utilization of microbiome modulators for human health benefits. However, the large-scale screening and optimization of modulators to validate microbiome modulation and predict related health benefits may be practically difficult due to the need for large number of animals and/or human subjects. To this end, in vitro or ex vivo models can facilitate preliminary screening of microbiome modulators. Herein, it is optimized and demonstrated an ex vivo fecal microbiota culture system that can be used for examining the effects of various interventions of gut microbiome modulators including probiotics, prebiotics and other food ingredients, aside from nutraceuticals and drugs, on the diversity and composition of the human gut microbiota. Inulin, one of the most widely studied prebiotic compounds and microbiome modulators, is used as an example here to examine its effect on the healthy fecal microbiota composition and its metabolic activities, such as fecal pH and the fecal levels of organic acids including lactate and short-chain fatty acids (SCFAs). The protocol may be useful for studies aimed at estimating the effects of different interventions of modulators on fecal microbiota profiles and at predicting their health impacts.

This protocol describes an in vitro batch-culture fermentation system of human fecal microbiota, using inulin (a well-known prebiotic and one of the most widely studied microbiota modulators) to demonstrate the use of this system in estimating effects of specific interventions on fecal microbiota composition and metabolic activities.

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

A culture from human stool for diagnosis of Campylobacter-based intestinal illness takes several days, a wait that taxes the fortitude of the physician and the patient. A culture is also prone to false negative results from random loss of viability during specimen handling, overgrowth of other fecal flora, and poor growth of several pathogenic Campylobacter species on traditional media. These problems can confound clinical decisions on patient treatment and have limited the field from answering fundamental questions on Campylobacter growth and infections. We describe a procedure that estimates the lower limit of bacterial numbers that can be detected by a culture and a method for quantifying survival of C. jejuni in media used for transport of this fragile organism. Knowing this information, it becomes possible to set clinically relevant detection thresholds for diagnostic tests and address unstudied issues of whether non-symptomatic colonization is prevalent, if co-infection with other enteric pathogens is common, or if bacterial load correlates with symptoms or serious sequelae. The study also included testing of 1,552 prospectively collected patient diarrheal fecal specimens that were initially classified by conventional culture and further tested by a new enzyme immunoassay. Positive and discrepant specimens were then screened by four molecular methods to assign true-positive or true-negative status. The 5 non-culture methods showed complete agreement on all 48 positive and discrepant specimens, while the culture mis-identified 14 (28%). The specimens that were incorrectly identified by culture included 13 false negative and 1 false positive sample. This basic protocol can be used with multiple Campylobacter spp. and will allow the numbers of Campylobacter bacteria that produce symptoms of gastroenteritis in humans to be determined and for prevalence rates to be updated.

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

Although stool culture for Campylobacter is imprecise, it is still considered the gold standard for identification. Methods to determine the limit of detection and survival in transport media of C. jejuni in human stool are described and compared with a new immunoassay with better accuracy.

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

Glomerular cell death is a pathological feature of myeloperoxidase anti neutrophil cytoplasmic antibody associated vasculitis (MPO-AAV). Extracellular deoxyribonucleic acid (ecDNA) is released during different forms of cell death including apoptosis, necrosis, necroptosis, neutrophil extracellular traps (NETs) and pyroptosis. Measurement of this cell death is time consuming with several different biomarkers required to identify the different biochemical forms of cell death. Measurement of ecDNA is generally conducted in serum and urine as a surrogate for renal damage, not in the actual target organ where the pathological injury occurs. The current difficulty in investigating ecDNA in the kidney is the lack of methods for formalin fixed paraffin embedded tissue (FFPE) both experimentally and in archived human kidney biopsies. This protocol provides a summary of the steps required to stain for ecDNA in FFPE tissue (both human and murine), quench autofluorescence and measure the ecDNA in the resulting images using a machine learning tool from the publicly available open source ImageJ plugin trainable Weka segmentation. Trainable Weka segmentation is applied to ecDNA within the glomeruli where the program learns to classify ecDNA. This classifier is applied to subsequent acquired kidney images, reducing the need for manual annotations of each individual image. The adaptability of the trainable Weka segmentation is demonstrated further in kidney tissue from experimental murine anti-MPO glomerulonephritis (GN), to identify NETs and ecMPO, common pathological contributors to anti-MPO GN. This method provides objective analysis of ecDNA in kidney tissue that demonstrates clearly the efficacy in which the trainable Weka segmentation program can distinguish ecDNA between healthy normal kidney tissue and diseased kidney tissue. This protocol can easily be adapted to identify ecDNA, NETs and ecMPO in other organs.

Extracellular DNA (ecDNA) released during cell death is proinflammatory and contributes to inflammation. Measurement of ecDNA at the site of injury can determine the efficacy of therapeutic treatment in the target organ. This protocol describes the use of a machine learning tool to automate measurement of ecDNA in kidney tissue.

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

With the development of microecology in recent years, the relationship between intestinal bacteria and inflammatory bowel disease (IBD) has attracted considerable attention. Accumulating evidence suggests that dysbiotic microbiota plays an active role in triggering or worsening the inflammatory process in IBD and that fecal microbiota transplantation (FMT) is an attractive therapeutic strategy since transferring a healthy microbiota to IBD patient could restore the appropriate host-microbiota communication. However, the molecular mechanisms are unclear, and the efficacy of FMT has not been very well established. Thus, further studies in animal models of IBD are necessary. In this method, we applied FMT from wild-type C57BL/6J mice to IL-10 deficient mice, a widely used mouse model of colitis. The study elaborates on collecting fecal pellets from the donor mice, making the fecal solution/suspension, administering the fecal solution, and monitoring the disease. We found that FMT significantly mitigated the cardiac impairment in IL-10 knockout mice, underlining its therapeutic potential for IBD management.

The interaction of genetic susceptibility, mucosal immunity, and intestinal microecological environment is involved in the pathogenesis of inflammatory bowel disease (IBD). In this study, we applied fecal microbiota transplantation to IL-10 deficient mice and investigated its impact on colonic inflammation and heart function.

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