Improved Method For Detection Of Methylation Status Of Genes From Limited, Archived, FFPE And FNAC Samples.

摘要

Purpose: Bisulphite modified genomic DNA and downstream analysis methods are the most powerful techniques which are used to determine the methylation of chromosomal DNA and the promoter region. However, the amount of material available is the most limiting factor, which continuously leads to the development of the most sensitive and specific method of methylation determination. In the present communication, we present an improved modification of bisulphite conversion and MSP.Method: Our strategy is the bisulphite conversion of direct tissue sections in the tube, followed by DNA purification and methylation specific PCR.Results: Our results successfully yielded a high amount of methylated DNA and showed promoter methylation amplification using very scanty biopsy sample, other clinical FFPE tissues and FNAC cells. A large no of genes could be studied, which otherwise would not be feasible using the conventional method of DNA isolation and bisulphite modification.Conclusion: Our method improves substantially, the previously published protocol in terms of yield, quality using a limited amount of tissue from formalin fixed material and cytology smears from fine needle aspirates.Abbreviations: sFRP1: secreted frizzled-related proteins 1, MGMT (O6-methylguanine-DNA methyltransferase), FFPE: Formalin fixed paraffin embedded, FNA: Fine needle aspirate, MSP: methylation specific PCR