Figure 5

Characterization of experimental errors in eMLPA and qPCR. (A) Correlation of eMLPA and qPCR. To verify eMLPA, data obtained from samples analyzed with both qPCR and eMLPA are plotted against each other. (B) Inter-experimental variation in heterozygous control (Pkd1^{del2-11, lox}) mice is presented. Raw data from five different eMLPA or qPCR experiments were combined. The percentage of Pkd1 deletion, which should be exactly 50%, was calculated relative to the median of all measurements. An eMLPA experiment consists of two or three hybridizations on which a single PCR was performed and a qPCR experiment consists of a triplo PCR. Each point shown is an average of these duplo or triplo measurements and the error bars represent the standard error of the mean. (C) The performance of eMLPA at low DNA concentrations. Single hybridizations were carried out on 500 ng, 25 ng or 10 ng of DNA from eight Pkd1^{del2-11, lox} mice, followed by extension, ligation and 29, 33 or 35 cycles of amplification respectively. Pkd1^del2-11 percentages were calculated relative to the median from the 500 ng hybridizations which was used as the 50% reference. From the eight Pkd1^{del2-11, lox} samples in each group; the 500 ng, 25 ng and the 10 ng hybridizations, the median and standard deviations from the Pkd1^del2-11 percentages are shown. Whereas the median from both groups at the lower DNA concentrations is close to the expected 50%, the variation in these groups is higher.