University of Liverpool, Liverpool, England;
University of Liverpool Cancer Research Centre, Roy Castle Lung Cancer Research;
University Hospital Aintree, Liverpool
Background: Promoter methylation of tumor suppressor genes has been investigated by a variety of means, recently including pyrosequencing.
Methods: Fresh tumor tissue and normal tissue from resection margin were obtained from 79 patients undergoing resection for squamous cell carcinoma. DNA was extracted and bisulphite treated. Polymerase chain reaction primers were designed to amplify 75– to 200–base pair regions of the CpG-rich gene promoters of p16, RAR-beta, E-Cad, CYGB, cyclin A1, MGMT, ATM, hMLH1, STAT1, and TIMP3. Methylation status of 5 to 22 individual CpG sites per gene was determined by pyrosequencing. Reverse transcriptase–PCR was used to correlate these data with mRNA expression.
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