High-Resolution Quantitative Methylation Analysis of MicroRNA Genes Using Pyrosequencing
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MicroRNA (miRNA) genes have been shown to perform a crucial role in breast cancer metastasis. The epigenetic inactivation of such microRNA genes, as a result of aberrant DNA methylation, is frequently found in human tumours including those of the breast, and this is an area of considerable research activity.
Pyrosequencing™ is a new quantitative method for the assessment of DNA methylation, with single CpG site resolution. Pyrosequencing™ can easily be performed in a 96-well-plate format with a cost-effective medium-sized throughput.
This chapter provides a general outline of DNA methylation analysis, a detailed protocol of the Pyrosequencing™ procedure, and guidelines for the design of new assays. The strengths and limitations of this approach are discussed throughout the chapter.
