Abstract
Epigenetic regulation of gene expression by DNA methylation is a central mechanism governing the silencing of tumor suppressor genes in many forms of cancer. Current methods have not proven optimal for the quantitative analysis of DNA methylation and corresponding in situ protein expression within cells in small specimens like skin biopsies. We have overcome this limitation by combining and modifying several techniques: target cell enrichment, DNA micro-isolation, one-step denaturation/bisulphite conversion/in-column desulphonation, specially designed PCR amplification, pyrosequencing and multispectral image analysis. Using this approach optimized for small samples, we can quantify minor alterations in gene methylation and protein expression using minimal amounts of tissue. Comparative studies of fresh and processed cells showed that our method is valid for DNA in both fresh and formalin-fixed, paraffin-embedded specimens. We can measure the effects of DNA methylation inhibitors, administered in vitro or in vivo, on the promoter methylation and protein expression of selected genes in specific cells. This novel approach should prove useful for a wide variety of investigative and clinical applications in dermatology and other specialties where the collection of small, routinely processed biopsy specimens is common. We refer to this method as Q-GAME (quantitative gene analysis of methylation and expression).