Development of a Quantitative Methylation-Specific Polymerase Chain Reaction Method for Monitoring Beta Cell Death in Type 1 Diabetes
Figure 2
Rationale for selection of the primers that differentiate between methylated and unmethylated CpG.
A) Schematic illustration of the mouse Ins2 gene with promoter region (blue), exon 1 (yellow), intron 1 (white), and exon 2 (green) showing the positions of CpG sites and the primers used in this study. Black arrows represent the bisulfite-specific primers (BSP) that amplify both methylated and unmethylated DNA. Red arrows represent methylation-specific primers (MSP) that amplify unmethylated but not methylated DNA. B) Gel electrophoresis (3% agarose) of PCR products amplified by reactions using different primer sets and the cloned Ins2 gene as template. The clone was methylated (M) or sham methylated (N) and bisulfite-treated prior to use in the reactions. NTC means non-template control. TSS indicates the transcription starting site.