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		The Real-Time TaqMan PCR and Applications in Veterinary Medicine



		· Introduction · From PacMan to TaqMan - a computer game revisited · The advantages of real-time TaqMan PCR over conventional quantitative PCR · Applications in Veterinary Medicine · Allelic discrimination · Discussion · The veterinarian and his relationship with the next-generation PCR technology · Acknowledgements · References
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Quantification of gene expression
RT-PCR is the technique of choice for analysing extremely low abundance mRNA derived from cells or tissues. PCR is a well established method, the sensitivity of which is a principal advantage over other techniques, such as Northern blots or RNase protection assay. Once again, the competitive approach is the most commonly used in this field; it ensures normalisation of differences in the kinetics of the reverse transcription reaction by using an internal control, known as the competitor. In due course, the real-time TaqMan PCR will replace many of the conventional systems because of these advantages. We have established real-time TaqMan systems for quantification of gene expression in different species. Inevitably, these assays have received great interest for their use in veterinary research and have been introduced into a number of different projects. Three ongoing veterinary research projects have been chosen to exemplify the usefulness of quantification of gene expression using real-time TaqMan PCR systems.

Cytokines play a central role in the regulation of cell differentiation, proliferation and cell-cell communication [5]. In addition, some cytokines have important effector functions via activation of cytotoxic compounds (eg. perforin, oxygen and nitric oxide radicals). Therefore, these hormones of the immune system are important for the definition of correlates of protective immunity, evoked by viral and bacterial infections or by vaccines. Cytokine induction in cats immunized with an experimental FIV DNA subunit vaccine, which used feline IL-12 as an adjuvant, has shown the involvement of IL-12 p40, IFN-g, and IL-10. Up regulation of mRNA for these cytokines was observed in cats with complete protection against a FIV challenge infection but not in control cats immunized with a placebo vaccine, consisting of uncoated gold particles and FIV gp140 coated with gold particles [35] (Figure 4).

Fig.4 Transcription of cytokines after immunising cats with a DNA vaccine. Cytokine transcriptions are shown 8 weeks after FIV infection challenge. Each cytokine signal is normalised against the GAPDH signal and then calibrated against the lowest normalised cytokine signal at 8 weeks. Bars represent ± standard error (SE) of normalised cytokine signals of 4 cats per vaccine group and control group.
* Cytokine transcription of cats from the vaccine group, FIV gp140, given IL-12 as the adjuvant, differed significantly from transcription of control cats (PMWU<0.05).

DNA and RNA extraction from archived formalin-fixed and paraffin-embedded (FFPE) tissues are of considerable interest for retrospective studies. Embedding bone and other calcified tissues in paraffin requires prior decalcification, a process that takes several weeks or months to complete and increases the risk of RNA degradation. As an alternative, bone and calcified tissues may be embedded in a hydrophobic acrylic resin, based on methyl methacrylate (MMA) [10]. The embedding process is completed within three weeks and does not involve a long process of decalcification. These final sections demonstrate excellent morphological preservation and are ideal for computerised image analysis. Furthermore, in situ hybridization has demonstrated that cellular RNA is still accessible for hybridisation [13]. While DNA extracted from FFPE tissue has been used successfully for many applications, reports describing the analysis of RNA extracted from FFPE tissues or MMA embedded tissues have been rare, or even absent [16,30,56,57].