Fig. 5

Multiplex GR bisulfite sequencing assay detection limit. T47-D (GR-methylated) DNA was added in varying amounts (10 to 0.01 ng) to a fixed amount (10 ng) of MCF-7 (GR-unmethylated) DNA. These mixtures (50%, 10%, 1%, and 0.1% T47-D DNA) and samples containing T47-D only (100%) and no T47-D DNA (0%, MCF-7 DNA only) were used to test the ability of the multiplex bisulfite sequencing assay to detect GR-methylated DNA in a background of unmethylated DNA. DNA from MCF-7 cells was assayed multiple times and was used to set a threshold for methylation which was defined as three standard deviations above the mean methylation level of all GR bisulfite sequencing regions (0.015, indicated by a dotted line). The primer sets for regions C, D, and U could detect as little as 1% T47-D DNA with this cutoff, and all could detect the sample with 10%