Examining the Vector–Host–Pathogen Interface With Quantitative Molecular Tools - Bacterial Pathogenesis: Methods and Protocols - page 123

4. Notes
1. Primer Express software v2.0 was used to select TaqMan primer and probe
sequences for the Y. pestis pla gene.
2. The PCR reaction can be optimized by empirically predetermining the annealing
temperature and MgCl 2 concentration that results in the minimum C t values.
3. Skin biopsy samples can be minced or homogenized with a tissue grinder before
DNA extraction.
4. Sensitivity of qPCR may also be improved by diluting the DNA template purified
from skin samples, which may contain PCR inhibitors (19) . Prior to use as qPCR
template, we routinely diluted DNA extracts from skin samples 1:10, 1:50, or
1:100 in DNA resuspension buffer, depending on the weight (<5, 5-7, or 7-10
mg, respectively) of the original skin biopsy.
5. Mechanical disruption of fleas using a bead mill is an alternative to manual
titration using a pestle (20) .
6. The limit of detection from skin biopsy samples was 10 3 Y. pestis . The use of
reverse transcriptase qPCR of a highly expressed gene may result in greater
sensitivity (21) .
7. The limit of detection from lymph node samples was also 10 3 Y. pestis . However,
only one-tenth of the sample was used for qPCR; the majority of the sample was
used for other experimental purposes. Extracting DNA from the entire sample
would increase the sensitivity.
8. We used the Puregene cell, tissue, body fluid, and Gram-negative bacteria DNA
purification kit from Gentra for mouse skin and flea samples. The manufacturer's
protocol was used with the following modification for use with skin samples:
following proteinase K and RNase treatment, the samples were passed through
a Qiashredder column to fragment the DNA and to remove mouse hair.
9. We used the Qiaprep spin miniprep kit from Qiagen for lymph node samples.
This extraction method enriches for bacterial plasmid DNA, which may be an
advantage because the pla gene target is on a Y. pestis -specific plasmid.
10. Sample collection and treatment can be tailored to fit additional experimental
requirements. For example, we collected lymph node contents in RNA protect
reagent, because part of the sample was used for gene expression analysis and
part for qPCR.
References
1. Dye, C. (1992) The analysis of parasite transmission by bloodsucking insects.
Annu. Rev. Entomol . 37 , 1-19.
2. Bell, A.S. and Ranford-Cartwright, L.C. (2002) Real-time quantitative PCR in
parasitology. Trends Parasitol . 18 , 337-342.
3. Edwards, K., Logan, J. and Saunders, N. (eds.) (2004) Real-Time PCR. An Essential
Guide . Horizon Bioscience, Norfolk, UK.
4. Kochanowski, B. and Reischl, U. (eds.) (1999) Quantitative PCR Protocols .
Humana Press, Totowa, NJ.