Figure 5

Analysis of the amplified/duplicated chromosomes. (A) Schematic of the amplified/duplicated chromosome 8q. The methylation levels for the selected genes in the 38 sporadic renal cell carcinomas (RCCs) are represented by green ovals for no significant methylation or yellow (β-values ≥0.33) and red (β-values ≥0.4) ovals for positive levels of hypermethylation. The methylation levels for the nine normal kidneys are represented by green ovals for no methylation (β-values <0.25). The degree of re-expression after 5′-aza treatment in the 11 RCC cell lines is represented by green ovals for no significant increase/change or yellow ovals for a positive increase (4-fold or greater) and red ovals for a highly positive increase (8-fold or greater). Other genes of interest were mapped to the chromosome, with red arrows representing oncogenes, black arrows representing known hypermethylated RCC-associated genes and blue arrows representing genes known to be mutated in RCC. The blue star indicates the gene is both mutated and hypermethylated. (B) Example reverse transcription (RT)-PCR analysis of the tumour/associated kidney normal pairs for all the selected genes for chromosome 8q and (C) the positive gene on chromosome 7. Each pair for each gene is shown with the tumour result on the left and the associated normal on the right and with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) band to demonstrate loading. Generally, the loading was skewed so that more tumour cDNA was analysed than the associated normal cDNA to emphasise that any loss observed was real and significant. In all cases at least one pair was shown where no loss/decrease was observed to demonstrate the mRNA would normally be expressed in the tumour tissue.