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Real-Time TaqMan PCR and Applications in Veterinary Medicine
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Because of this situation, we tested the feasibility of quantitative real-time TaqMan RT-PCR in MMA-embedded compared to fresh samples. Biopsies of subchondral cystic lesions (SCL) were taken from horses either during arthroscopic surgery or post mortem; these were fixed in paraformaldehyde and embedded in MMA. Though RNA extracted from MMA embedded bone sections was severely degraded, it could be reverse transcribed with random hexamer primers and amplified, using real-time TaqMan PCR systems for the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the proinflammatory cytokines interleukin (IL) 1-b and IL-6 and the inducible form of NO synthase (iNOS) (Figure 5), which show distinct differences of gene expression in different areas of the SCL34. Analysis of MMA-embedded tissues with real-time TaqMan PCR allows an additional refinement of the method. Tissue structure is preserved excellently in MMA blocks, and sawed sections from these blocks can be dissected further to allow localised measurements of gene expression.    Fig.5 Quantitative mRNA analysis of IL-1b, iNOS and IL-6, all normalised to mRNA of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). 50-micrometer sections from methyl methacrylate (MMA) embedded tissues were cut on a sawing microtome. Specific areas of the subchondral cystic lesion (SCL) were excised using a micro-scissor; total RNA was extracted and reverse transcribed into cDNA for further analysis and quantification of mRNA for IL-1b, iNOS and IL-6. The signal of GAPDH mRNA was used to normalise for differences in RNA extractions and for different efficiencies of cDNA synthesis. An understanding of immune regulation during infections of the mammary gland in dairy cattle is important for the design of prophylactic vaccines and for the optimization of therapeutic protocols. Using real-time TaqMan PCR, we addressed gene transcription in milk cells of Holstein cows during mid-lactation to define the cytokine profile of these cells in a healthy mammary gland [37]. Cows selected for this study showed relatively low somatic cell counts of <105/ml milk, however, transcription of IL-8, TNF-a, IFN-g, and GM-CSF mRNA was detected in all samples and IL-12 p40 in 6 of 7 samples (Figure 6). Holstein cows are bred for high milk production causing a permanent state of stress in these animals. As a result, a certain amount of tissue damage and possibly low-grade bacterial presence is common. The assumption of the apparent healthy state of the animals is important in order to understand the expression of cytokines indicating a certain level of inflammation. In addition, this study revealed differences between cytokine profiles determined with conventional quantitative RT-PCR and with real-time TaqMan PCR systems. Real-time TaqMan PCR systems are a promising alternative to conventional quantitative PCR systems and could provide a better understanding of immuneregulatory cytokines and their potential use for diagnosis, prophylaxis and immunotherapy.  Fig.6 Upregulation in transcription was measured for six different cytokines present in milk cells collected in mid-lactation from seven healthy Holstein cows. Bars represent the means of cytokine transcription. Cytokine transcription is expressed as n-fold differences to the calibrator, which was the lowest detectable cytokine signal. Note the semilogarithmic scale.
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