Figure 4

X chromosome inactivation analysis by HUMARA assay shows a prevalent polyclonal pattern of Malignant Mesotheliomas. Gel electrophoresis (A). PCR products from mock digested (H-) and HpaII-digested (H+) DNA samples were separated on a 3% agarose gel and visualized under UV light, using ethidium bromide. Capillary-Electrophoresis (B, C). HUMARA PCR assay was performed using a 5FAM-labeled forward primer, and quantified by the Applied Biosystems 3100 Genetic Analyzer. Two major peaks denote the two allelic HUMARA loci PCR-amplified in HpaII-digested (H+) and mock-digested samples (H-). The allele intensities were measured as peak area of both alleles, which is proportional to the molar amount of DNA. Peak areas were calculated for each allele using Genescan software, as described in the Material and Methods. A CR ≥3.0 or ≤0.33, representing a preferential loss of intensity in the digested sample of one of the two alleles present in the tumor sample, was scored as a monoclonal pattern.