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

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.