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The primers used for amplification of methylated and unmethylated promoters of genes RARB , TIMP3 , CDKN2A , p14 ARF , MGMT , DAPK , CDH1 , GSTP1 , APC promoter 1A, RB1 , MLH1 , TP73 , BRCA1 , FHIT , and HIC1 have been described previously http://pathology2.jhu.edu/pancreas/prim0425.htm#MSP; [ 32 47 48 49 ] . Methylated (MF and MR) and unmethylated (UF and UR) primers for RASSF1A promoter were designed according to Herman et al [ 46 ] and as described earlier [ 50 ] . PCR products were run on 2% agarose gels and visualized after ethidium bromide staining.
The primers used for methylated and unmethylated-specific PCR for genes RARB , TIMP3 , CDKN2A , p14 ARF , MGMT , DAPK , CDH1 , GSTP1 , APC promoter 1A, RB1 , MLH1 , TP73 , BRCA1 , FHIT , and HIC1 have been described previously http://pathology2.jhu.edu/pancreas/prim0425.htm#MSP; [ 34 35 36 37 ] . For additional genes, we designed the following gene-specific primers for methylated (MF and MR) and unmethylated (UF and UR) sequences according to Herman et al [ 33 ] :