Figure 3

Hypermethylation analysis by bisulfite sequencing, MSP and MCA-Meth of MGMT gene in astrocytoma samples 26, 28 and in the U87MG cell line. a) Bisulfite sequencing results of the MGMT gene in astrocytoma samples 26 and 28 and in the U87MG cell line. Black squares indicate methylated CpG dinucleotides and white squares indicate unmethylated CpG dinucleotides in promoter sequences. Above the diagrams, the area of the promoter analyzed by MSP, MCA-Meth and bisulfite sequencing is indicated. The 5'- and 3'-priming sites of M- and U-primers are indicated as small black dots representing the CpG sites incorporated in the primer sequence. The position of CpGs in relation to the CpG-island as well as the number of CpGs analyzed are indicated in the brackets behind the respective techniques. On the right, the diagrams of peaks obtained with the sequencing software: it can be observed how after the bisulfite treatment, the cytosines remain unchanged in the methylated sample, while there is a shift to thymine in the unmethylated sample. b) MSP results of MGMT in astrocytoma samples 26, 28 and in the U87MG cell line. U: PCR with primers specific for the unmethylated sequence; M: PCR with primers specific for the methylated sequence. c) MCA-Meth results of MGMT in astrocytoma samples 26, 28 and in the U87MG cell line. The graphs show the unmethylated control peak (blood DNA) in red, the methylated control peak in green, the no template control in blue and the sample or cell line peak, in brown. It can be observed how in the case of the U87MG cell line, the amount of methylated cytosines in the area of the promoter analyzed by bisulfite sequencing is intermediate between the sample 26 (unmethylated) and sample 28 (fully methylated). Of note, MCA-Meth appreciates this inhomogeneous sequence methylation pattern of U87MG with a melting curve peak in an intermediate position as compared to the unmethylated (26) and the methylated sample (28).