Examining the Vector–Host–Pathogen Interface With Quantitative Molecular Tools - Bacterial Pathogenesis: Methods and Protocols - page 122

3.2.2. Skin Biopsies of Fleabite Site
1. After a flea has attempted to feed on a mouse, euthanize the animal, wipe the
area with an alcohol pad, and aseptically collect a skin biopsy of the bite site.
2. Place the biopsy in a 1.5-mL microfuge tube.
3.2.3. Lymph Nodes from Infected Rats
1. Aseptically dissect lymph nodes from euthanized, infected rats.
2. Immediately after dissection, disrupt each lymph node by pressing it through a
70-μm cell strainer into 10 mL of RNA protect.
3. Transfer one-tenth (1 mL) of the lymph node suspension to a 1.5-mL microfuge
tube and centrifuge at 13.5 × g for 5 min. Remove and discard the RNA protect
supernatant.
3.3. DNA Extraction
1. A number of commercially available DNA extraction kits are suitable to prepare
purified total DNA from tissue samples. The method chosen can be based on
individual preference, sample characteristics (e.g., ratio of microbial to host cells),
qPCR target, and specific experimental needs ( see Notes ).
2. Assess purity and quantity of purified DNA by A 260 /A 280 spectrophotometry.
3.4. Real-time qPCR
1. Prepare a sufficient volume of 1× TaqMan master mix containing 500 nM of
forward and reverse primers and 200 nM of probe to perform qPCR on each
control, standard, and unknown sample in triplicate. The total volume of individual
TaqMan reactions is 25 μL for the ABI Prism 7700 and 20 μL for 7900HT.
2. Pipet master mix for individual reactions to wells of the 96-well reaction plate.
3. Add 1-5 μL of template DNA prepared from negative control, standard, and
unknown samples. Do not add any template to three “no-template” control wells.
4. For the Y. pestis pla gene primer and probe set and the ABI 7700 instrument, the
reaction program consists of 95 °C for 10 min followed by 45 cycles of 94 °C for 20
s, 60 °C for 20 s, and 72 °C for 30 s. For the 7900HT instrument, the program is 50 °C
for 2 min, 95 °C for 10 min, and then 40 cycles of 95 °C for 15 s and 60 °C for 1 min.
3.5. Data Analysis
1. Using the software package supplied with the TaqMan instrument, generate a
standard curve by plotting the C t values of the standards on the y -axis and the
log10 number of Y. pestis cells they contained on the x -axis.
2. Determine the number of Y. pestis present in the unknown samples by using their
C t value in the linear equation of the standard curve to solve for x , the log number
of Y. pestis .