Quantitative measurement of KDR and VE-cadherin gene expression in MM using real-time RT-PCR. The cDNA of PBMCs from patients with MM or controls was used to amplify KDR, VE-cadherin, and GAPDH in real-time PCR reactions. An optimized threshold line was determined in the range of linear amplification to compare cycle number in different samples. The expression of KDR and VE-CADHERIN was calculated by the comparative threshold cycle method. Reproducibility of this method was confirmed by repetition of each experiment once using frozen aliquots of the same mRNA extracted within 6 hours of collection of PBMCs from subjects and stored at -20°C for 4 weeks. (A) Representative amplification plots for KDR (left) or VE-cadherin (right) expression in PBMCs from an MM patient and a control subject; GAPDH amplification was used as an internal control. While GAPDH levels for both MM and control are comparable, indicating the same amount of cDNA used in each reaction, expression of KDR (C_T = 30.2 for control, C_T = 26.3 for MM) and VE-cadherin (C_T = 32.9 for control, C_T = 31.1 for MM) were different. Arrows indicate the C_T values. (B) Relative mRNA expression of KDR is shown for patients (filled circles) and controls (open circles). (C) KDR mRNA level is positively correlated with concurrently determined serum M protein levels. The association between KDR mRNA and M protein was evaluated by Pearson r.