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Materials and methods
Real-time polymerase chain reaction
Real-time quantitative polymerase chain reaction (PCR) was carried out using the LightCycler PCR system (Roche Diagnostics, Meylan, France) using the DNA binding SYBR Green I dye to detect the PCR products as described previously [19]. A serial dilution was used to generate each standard curve. Each 20-μL reaction mixture contained 1× LightCycler-DNA master SYBR Green I, a specific primer, and 2 μL of cDNA. After 2 minutes of denaturation at 95°C, the MMPs, VEGF, IL-6, IL-8, cyclooxygenase-2 (COX-2), and β-actin underwent 40 reaction cycles at 95°C for 5 seconds, 55°C to 60°C for 10 seconds (annealing), and 72°C for 13 seconds. Product specificity was determined by melting curve analysis as described in the LightCycler manual. The results are calculated as ratios of gene transcripts to β-actin transcripts, with the quantity of transcripts in each sample expressed as a copy number. The ratio of MMPs, VEGF, IL-6, IL-8, and COX-2/β-actin mRNA was assigned a value of 100%, with the corresponding results calculated as relative percentages. The primers were synthesized by Bioneer Co. Ltd. (Seoul, Korea), and their sequences are listed in Table 1.

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