Fig. 3

The Figure shows a schematic representation of a theoretical rationale for MSP results based on previous works [23, 28, 29]. The DNA templates (long bars with 3′- > 5′ directionality designation) show blue and red dots representing unmethylated and methylated CpGs, respectively. The short blue and red bars represent unmethylated- and methylated-specific primer pairs, respectively. For simplicity, we show methylation patterns with uniform methylation status at the primer’s annealing site on DNA template (a-h) and only two with non-uniform methylation status (i and j). While fully unmethylated (a) and fully methylated (b) patterns provide unmethylated- and methylated-specific positive PCRs, respectively, partially methylated patterns (c-j) can have any of the four possible qualitative MSP results. MSP-inexpressible partially methylated pattern (d) may rationalize our doubly negative malignant sample (Sertoli-Leydig cell tumor), in addition to possible mutation as methylated CpGs are known hotspot for mutations [39, 40]