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제안 방법
- The information in this study should help investigators implement qRT-PCR assays for the cytokine genes quantified here, without having to repeat the laborious validation process, as long as the conditions validated by our trials are maintained. In addition, this study outlines the procedures and techniques for developing a straightforward validation system for establishing primers to be used in qRT-PCR assays.
- Development of a SYBR green real-time PCR assay for canine cytokine gene expression was successfully achieved by proper primer design, annealing temperature and melt curve analysis. The information in this study should help investigators implement qRT-PCR assays for the cytokine genes quantified here, without having to repeat the laborious validation process, as long as the conditions validated by our trials are maintained. In addition, this study outlines the procedures and techniques for developing a straightforward validation system for establishing primers to be used in qRT-PCR assays.
- Assays for the detection of a single gene require the careful choice of primers, target sequence, and method for detecting the amplified products. This study describes the steps used to optimize and validate a reliable set of primer pairs for qRT-PCR assays using the fluorogenic DNA binding dye SYBR green. SYBR green chemistry was used in this study instead of Taqman probe chemistry, even though the latter may have given a higher specificity (6).
- SYBR green chemistry was used in this study instead of Taqman probe chemistry, even though the latter may have given a higher specificity (6). Unlike SYBR green methods, designing assays using TaqMan probe chemistry is not always possible, because this technique requires that the primers and probes satisfy rigid constraints that are not always easily achieved, especially with the gene sequences selected in this study. Using SYBR green techniques, however, we obtained highly reliable and accurate quantification of canine cytokine gene expression, using properly validated methods.
이론/모형
- Cell concentration and viability were assessed using an impedance cell counter and cells suspended in trypan blue solution, respectively. Cell purity was determined using a conventional Diff-Quik method after Cytospin-centrifuge preparation. Viability was demonstrated to be high at > 98%, and > 95% of cells were PBMCs.
- The comparative CT method can be used only if the amplification kinetics of the target and housekeeping genes are approximately equal (7), as determined by a validation experiment. If the efficiencies of the two amplicons are not approximately equal, then the analysis should be performed using an absolute quantification method with standard curves. However, this method requires a standard template source, such as purified cloned cDNA, or PCR-amplified cDNA that must included in each run, limiting the number of samples that can be assayed on the same plate.
- Primers were re-designed if any regions of the templates were predicted to form secondary structures above the annealing temperature of the PCR reaction. Primer uniqueness and specificity were verified using the Primer-Blast Tool (http://www. ncbi.nlm.nih.gov/tools/primer-blast) using GenBank Accession numbers. Primers were commercially synthesized by the Genotech Corporation (Daejeon, Korea).
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