Efficiency of quantitative simplex (QS) and multiplex (QM) methylation-specific PCR. The diagrams illustrate comparison of both methods, namely A: quantitative multiplex methylation-specific PCR (QM-MSP) and B: quantitative singleplex methylation-specific PCR (QS-MSP). The reactions have been performed in duplicate. We used a mixture of primers and hydrolysis methylated probe specific to only amplify methylated alleles of Albumin, WIF1, PENK, and NPY genes along with a titration of human genomic DNA at various concentrations ranging from 10 up to 0.01 ng/well. On each dilution, the cycle threshold (Ct) was determined for standard DNA. Nearly identical Ct values for each DNA dilution indicate uniform primer performance over 3 logs. The slope of −3.32 (100% efficiency) reflects a 2-fold amplification of DNA per cycle corresponding to a high efficiency. The correlation coefficient R2 of 0.99 shows a high degree of linearity over the entire range.