2. DNA Extractions
To consistently extract microbial DNA from our avian fecal and preen oil samples, we revised the original Applied Biosystems™ PrepMan™ Ultra Sample Preparation Reagent method for extractions from bacterial samples, adding a bead beating step. For our fecal samples, we first removed approximately 0.15 g feces from each sample, taking care to avoid collecting any of the white uric acid. After pouring any ethanol off the fecal sample, we placed the sample in a weigh boat in a fume hood for about one minute to allow the evaporation of excess ethanol. We then transferred the fecal sample into a 2 mL Fisherbrand™ Free-Standing Microcentrifuge Tube with Screw Cap that had been filled with approximately 100 µL of 460 nm acid-washed glass beads (Sigma-Aldrich, Inc., St. Louis, Missouri, USA) and 200 µL of PrepMan™ Ultra Sample Preparation Reagent (Thermo Fisher Scientific, Waltham, Massachusetts, USA). For preen oil samples, we placed the entire capillary tube tip (containing the preen oil sample) into a 2 mL microcentrifuge tube containing the same volumes of acid-washed glass beads and PrepMan™ Ultra Sample Preparation Reagent as used with the fecal samples. Both types of sample mixtures were homogenized using a Mini-Beadbeater (BioSpec Products, Bartlesville, Oklahoma, USA) for 1 minute on high, and then placed in a water bath for 10 minutes at 100°C. For preen oil samples, we removed the capillary tube tip after the water bath step, at which point the oil sample was no longer observable in the tip of the capillary tube. For both sample types, we then centrifuged samples at 14,000 rpm for 2 minutes and pipetted off the supernatant. This supernatant was then used as the DNA extract for PCR. The volume of the final DNA extract samples was ~75µL, depending on how much could be pipetted off without collecting the acid-washed beads. To create a negative control, we followed this protocol using 100 uL of water instead of a fecal sample. For a positive control, a colony ofEscherichia coli was prepared and used for extraction and PCR amplification.
3. PCR Amplification of microbial 16S rRNA
To assess the success of our microbial DNA extraction method, we performed PCR amplification of hypervariable regions of the 16S rRNA gene (V3-V4 regions) using our DNA extracts from both fecal and preen oil samples. The 16S rRNA gene is commonly used to identify bacterial taxa and quantify microbial diversity. This gene is present in all bacteria and its highly conserved nature coupled with species-specific regions of variation allows for identification of different clades of bacteria.
For most of our DNA extractions from fecal samples, we first mixed 1 µL of the concentrated fecal DNA sample with 99 µL of nuclease free water. If the fecal sample used for microbial extraction was less than 0.1 g, we used a non-diluted DNA extract sample. Due to the small volume of collected preen oil samples, ~1-2 mg, we did not dilute the preen oil DNA extracts.
We used a total PCR volume of 20 uL, containing master mix, GC enhancer, forward and reverse 16S rRNA primers, DNA, and water. Specifically, each reaction included 10 uL Platinum™ II Hot-Start Green PCR Master Mix (2X) from Invitrogen (Waltham, Massachusetts), 4uL of the Platinum GC Enhancer included with this master mix, 2uL of nuclease free H20, and 2uL of our DNA extraction. To amplify the V3-V4 region of the 16S rRNA gene, we also included 1uL each of 25uM 341F (CCTACGGGNGGCWGCAG) and 806R (GACTCHVGGGTATCT-AATCC) 16S rRNA primers.
Optimized PCR conditions included an initial denaturation step at 95°C for 2 min, followed by 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 68°C for 1 min, and a final elongation at 68°C for 2 min. To confirm successful PCR amplification, we ran each sample on a 2% agarose gel at 140 V for 50 minutes and verified the presence of bands visually. The expected product size was ~430 bp. For any extractions that did not show a band on the first agarose gel, we either ran the same PCR product on an additional agarose gel, or we ran a new PCR reaction using the original DNA extract. We therefore did not extract DNA multiple times from any of our samples.