We appreciate the help from the Argonne National Laboratory and our collaborating bioinformaticians. This work is supported by various NIH and internal grants.

The Cx3cr1gfp/gfp locus of B6.129P2(Cg)-Cx3cr1tm1Litt/J mice was backcrossed to MRL/MpJ-Faslpr/J (MRL/lpr) for 10 generations to generate MRL/lpr-CX3CR1gfp/gfp mice. Genome screening using single nucleotide polymorphism (SNP) panels confirmed that the genetic background of newly generated mice was more than 97% identical to that of MRL/lpr. After that, mice were bred and maintained in a specific pathogen-free environment following the specific requi...

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Balanced gut microbiota can protect the human body from diseases. Once this balance is disrupted by external or internal triggers, consequences can be devastating. This method presents a way to analyze the dynamics of gut microbiota in murine models. The method is suitable not only for comparisons among groups but also for tracking the gut microbiota over time to better identify time-dependent factors disrupting the gut microbiota.
All the mice in the experiment must be handled in the same env...

Humans and bacteria have coexisted for a long time. They have established a codependent relationship with mutual beneficial effects that influences host immune responses in quantitative and qualitative ways1. Recent studies suggest an association between the gut microbiota composition and the pathogenesis of autoimmune diseases that include multiple sclerosis2, rheumatoid arthritis3, type 2 diabetes4, Inflammatory bowel disease5, and systemic lupus erythematosus (SLE)6. However, whether the gut microbiota is the main cause or a secondary effect of these autoimmune diseases is still unclear7. Potentially, gut microbiota could exacerbate the disease during the effector phase of autoimmune disorders or play a role in regulating the induction of these diseases8.
Intestinal dysbiosis has been reported in female lupus-prone MRL/Mp-Faslpr (MRL/lpr) mice, and gut microbiota changes with a significant depletion of Lactobacilli were observed9. When a mixture of five Lactobacillus strains was orally administered, lupus-like symptoms were largely attenuated in these mice, suggesting an essential role of microbiota in regulating SLE pathogenesis.
The following technique of DNA extraction allows to follow microbiota fluctuations and analyze them qualitatively and quantitively during the course of murine SLE-like disease in lupus-prone mice. Whether to examine the healthy gut microbiota or define dysbiosis, it is important to critically examine how data is collected and whether it is accurate and reproducible10. Every step is critical in this process. An appropriate methodology must be used to extract microbial DNA as any possible problem introducing biases during the DNA extraction process could result in inaccurate microbial representation. While the phenol-chloroform method is described here, there are commercially available kits to extract DNA from bacteria that work well in particular cases11. However, their usability is limited by the cost and required sample quantity.
The protocol presented here is cost-effective and requires only a small amount of sample. It works fine with any kind of stool sample and is useful in studying the dynamics of the gut microbiota over time as well as comparisons among groups. DNA is isolated with a method of alcohol purification, which uses phenol, chloroform, and isoamyl alcohol. Alcohol-based extraction helps to clean and remove the sample of proteins and lipids, where DNA is precipitated in the final step. The proposed method has significantly high efficiency and quality and has been proven to be accurate in identifying bacterial populations. One critical note during the procedure is that DNA contamination can occur, and thus appropriate sample handling is required12.
The DNA is then analyzed by the Next Generation Sequencing platforms for the 16S rRNA gene, such as the Illumina MiSeq. In particular, the V4 hyper-variable region is analyzed to provide a better quantification for high-rank taxa13. The subsequent bioinformatics analysis is outsourced, followed by in-house analysis using standard statistical methods. There are numerous open-source bioinformatics software programs available for downstream sequencing, and the type of analyses performed depends heavily on the specific biological question of interest14. This protocol focuses specifically on the experimental steps prior to sequencing and provides a more versatile, cost-effective, comparable, and efficient method to obtain DNA from fecal samples.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.1 mm glass beads</td>
			<td>BioSpec Products</td>
			<td>11079101</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL screw cap tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td>3488</td>
			<td></td>
		</tr>
		<tr>
			<td>20% SDS</td>
			<td>FisherScientific</td>
			<td>BP1311-1</td>
			<td>SDS 20%</td>
		</tr>
		<tr>
			<td>96% Ethanol, Molecular Biology Grade</td>
			<td>Thermo Fisher Scientific</td>
			<td>T032021000CS</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Acetate (5 M)</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM9071</td>
			<td>NH4AC 5M</td>
		</tr>
		<tr>
			<td>B6.129P2(Cg)-Cx3cr1tm1Litt/J</td>
			<td>Jackson Laboratory</td>
			<td>005582</td>
			<td></td>
		</tr>
		<tr>
			<td>Bullet Blender storm 24</td>
			<td>Next Advance</td>
			<td>4116-BBY24M</td>
			<td>Homogenizer</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>FisherScientific</td>
			<td>C298-500</td>
			<td></td>
		</tr>
		<tr>
			<td>DEPC-Treated Water</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM9916</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediamine Tetraacetic Acid</td>
			<td>FisherScientific</td>
			<td>BP118-500</td>
			<td>EDTA</td>
		</tr>
		<tr>
			<td>Foil plate seal</td>
			<td>FisherScientific</td>
			<td>NC0302491</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes-Kimtech 34256</td>
			<td>FisherScientific</td>
			<td>06-666C</td>
			<td></td>
		</tr>
		<tr>
			<td>MRL/MpJ-Faslpr/J (MRL/lpr) mice</td>
			<td>Jackson Laboratory</td>
			<td>000485</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodrop 2000 spectrophotomer</td>
			<td>Thermo Fisher Scientific</td>
			<td>ND2000CLAPTOP</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol: chloroform: isoamylalchohol (25:24:1)</td>
			<td>FisherScientific</td>
			<td>BP1752I-400</td>
			<td>PCI</td>
		</tr>
		<tr>
			<td>Scale with 4 decimals</td>
			<td>Mettler Toledo</td>
			<td>MS205DU</td>
			<td></td>
		</tr>
		<tr>
			<td>Skirted 96-well plates</td>
			<td>Thermo Fisher Scientific</td>
			<td>AB-0800</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>FisherScientific</td>
			<td>15528154</td>
			<td>NaCl</td>
		</tr>
		<tr>
			<td>Tris Hydrochloride</td>
			<td>FisherScientific</td>
			<td>BP1757-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>Scientific Industries</td>
			<td>SI-0236</td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of fecal microbiota dynamics in lupus-prone mice using a simple, cost-effective dna isolation method

Gut microbiota has an important role in educating the immune system. This relationship is extremely important for understanding autoimmune diseases that are not only driven by genetic factors, but also environmental factors that can trigger the onset and/or worsen the disease course. A previously published study on the dynamics of the gut microbiota in lupus-prone MRL/lpr female mice showed how changes of the gut microbiota can alter disease progression. Here, a protocol is described for extracting representative samples from the gut microbiota for studies of autoimmunity. Microbiota samples are collected from the anus and processed, from which the DNA is extracted using a phenol-chloroform method and purified by alcohol precipitation. After PCR is performed, purified amplicons are sequenced using a Next Generation Sequencing platform at Argonne National Laboratory. Finally, the 16S ribosomal RNA gene sequencing data is analyzed. As an example, data obtained from gut microbiota comparisons of MRL/lpr mice with or without CX3CR1 are shown. Results showed significant differences in genera containing pathogenic bacteria such as those in the phylum Proteobacteria, as well as the genus Bifidobacterium, which is considered part of the healthy commensal microbiota. In summary, this simple, cost-effective DNA isolation method is reliable and can help the investigation of gut microbiota changes associated with autoimmune diseases.

The results from Argonne National Laboratory are analyzed by a qualified bioinformatician, followed by an in-house analysis of the data using standard statistical methods. Typical microbiome analyses involve clustering of similar sequences into operational taxonomic units (OTUs) or amplicon sequence variants (ASVs) as a proxy for different microorganisms in a sample. Counts of OTUs or ASVs across samples can then be used to test for changes in within-sample (alpha) diversity and between-sample (beta) diversity. They can ...

Watch this Scientific Journal Video about Analysis of Fecal Microbiota Dynamics in Lupus-Prone Mice Using a Simple, Cost-Effective DNA Isolation Method at JoVE.com

Analysis of Fecal Microbiota Dynamics in Lupus-Prone Mice Using a Simple, Cost-Effective DNA Isolation Method

This protocol provides a simple, cost-effective DNA isolation method for the analysis of murine gut microbiota alterations during the development of autoimmune disease.

Gut microbiota has an important role in educating the immune system. This relationship is extremely important for understanding autoimmune diseases that ...

The proposed DNA isolation method can serve to study the gut microbiota dynamics in autoimmune diseases mouse models. A more versatile, cost-effective, and efficient method is proposed to obtain DNA from fecal samples that are comparable to commercially-available kits. To begin, take each mouse individually out of its cage, collect a fecal pellet directly from the anus and store it at minus 80 degrees Celsius until being processed.Process the samples with sterile and clean forceps, tubes, and gloves. Add a frozen pellet to a pre-weighed, two milliliter screw cap tube, and record the fecal weight in grams, which should be between 0.02 to 0.05 grams per fecal pellet. Add to the pellet a thin layer of 0.1 millimeter glass beads, 500 microliters of lysis buffer, and 200 microliters of 20%sodium dodecyl sulfate.Bead beat the tube for four minutes in a homogenizer, followed by three to five minutes of vortexing. Centrifuge the tubes for a short time to eliminate the bubbles and foam. To extract DNA from the microbiota samples, transfer 350 microliters of sample supernatant to a new, 1.5 milliliters snap cap tube, without any debris.Add 500 microliters of phenol:chloroform:isoamyl alcohol, in a ratio of 25:24:1, and vortex for one minute. After centrifugation, transfer 180 microliters of the top aqueous phase into a new, 1.5 milliliter snap cap tube. Add 180 microliters of chloroform, invert and mix.From this, transfer 180 microliters of the aqueous phase into a new snap cap tube. In a biosafety cabinet, add 180 microliters of cold isopropanol and 36 microliters of five molar ammonium acetate. Invert it a few times to mix, and place the tube on ice for 20 minutes.Discard the supernatant, followed by washing the pellets several times with 500 microliters of cold 70%ethanol. Discard the supernatant, and air dry the tube upside down on tissue paper for 20 minutes, until the pellet becomes clear. Suspend the pellet in 50 microliters of molecular grade water, and heat the liquid at 37 degrees Celsius for 10 minutes to fully dissolve large DNA pellets.After heating, centrifuge these tubes at 3, 400 times G for one minute to recover the condensation droplets from the lid. Measure the concentration, and obtain the 260/280 ratio using a spectrophotometer. A ratio for good quality DNA is generally between 1.8 and two.Centrifuge the prepared samples at 600 times G for one minute at room temperature, before freezing at minus 80 degrees Celsius for 24 hours. Then, send 20 microliters of samples with concentrations ranging from one to 50 nanogram per microliters to Argonne National Laboratory for sequencing. The mouse strain lacking the receptor CX3CR1 has a different gut microbiota composition compared to wild-type MRL/lpr, MRL/lpr-CX3CR1 GFP/GFP mice, shows significant differences in certain genera, relevant to potentially pathogenic bacteria such as those in the phylum Proteobacteria.Be careful with contaminating the sample. It needs to be sterile. Also, it is important pipetting with accuracy when transferring the supernatant in a new tube.This method is suitable for molecular analysis such as PCR, restriction enzyme digestion, or genomic library construction. In lupus, we proved a strong relationship between the gut microbiota and lupus progression.

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Immunology and Infection

1.9K visualizações.  Virginia Polytechnic Institute and State University. O método de isolamento de DNA proposto pode servir para estudar a dinâmica da microbiota intestinal em modelos de camundongos com doenças autoimunes. Um método mais versátil, econômico e eficiente é proposto para obter DNA de amostras fecais que são comparáveis aos kits comercialmente disponíveis. Para começar, tire cada rato individualmente de sua gaiola, colete uma pelota fecal diretamente do ânus e armazene-a a menos 80 graus Celsius até ser processada.Processe as amost...