Fig. 2

Range of detection of GSTP1 promoter in DNA enriched by SEEMLIS. Methylated DNA was enriched by MBD2-MBD capture from DNA extracted from serially diluted LNCaP cells (n = 8 per dilution) and 1000 patient-derived (n = 10) or healthy donor (HD) (n = 18) white blood cells (WBCs). Quantitative PCR for GSTP1 and LINE1 was performed using enriched methylated DNA. A An ROC curve for all WBC samples and all LNCaP samples was created. Area under the curve (AUC) with 95% confidence interval is indicated. Optimal threshold (OT) values determined by Youden’s J statistic are listed with their associated sensitivity and specificity values. Detection limit was calculated using the slope of the best fit line of GSTP1 Ct values plotted against cell input. B MI was calculated by delta Ct relative to a max cycle value (MCV) of 45 with all undetected samples set to a Ct value of 45 for analysis. Optimal threshold as determined by ROC curve is shown as a dotted line. Each dot represents an individual sample taken from a pool of cells diluted to the indicated concentration. C LINE1 and GSTP1 Ct values were plotted against each other for all LNCaP samples. A simple linear regression was performed to determine the best fit line and 95% confidence interval for that line (shaded region). R and R² values are listed for the correlation. D GSTP1 Ct values were averaged for each cell input and plotted against the cell input values. A semi-log nonlinear fit was performed to determine the best fit line and the slope of the best of fit line (− 3.42). Each tenfold dilution of input should result in a gain of 3.32 Ct values, giving a slope of − 3.32 for a perfect assay (100% efficiency). E MI for serially diluted LNCaP cells spiked into 1000 WBCs from a patient (n = 8) is shown. Performed as described above for B. All error bars represent standard error of the mean (SEM)