Analysis of DNA methylation by pyrosequencing. Representative results of pyrosequencing showing analysis of 9 CpGs in the first exon of RASSF1A (from CpG6 to CpG14). The y-axis represents the signal intensity of luminescence in relative luminescence units (RLU) emitted following nucleotide base incorporation into the sequence, while the x-axis shows the nucleotide dispensation order, indicated by the letters below each graph: E, enzyme mix; S, substrate; A, G, C, T, nucleotides. The sequence in the upper left of the boxes is the region subjected to pyrosequencing analysis, where the nine letters Y indicate the respective cytosine in the 9 CpG sites (from CpG6 to CpG14) whose methylation status is shown here. Values in light blue boxes are the percentages of methylation of each CpG. Percentages of methylation represent the ratio between signal intensities of C and T in each C of a CpG site. Dispensations corresponding to the potentially methylated cytosine (C or T after bisulfite treatment) are highlighted in grey. Each panel depicts a representative sample showing no methylation (panel A, sample N1), partial methylation (panel B, sample T8) and high methylation (panel C, sample N2) levels.