High-resolution quantitative methylation analysis of microRNA genes using Pyrosequencing™.
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.
Institute of Pathology, Medizinische Hochschule Hannover, Hannover, Germany. Lehmann.Ulrich@MH-Hannover.de
Ulrich
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Lehmann
Cord
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Albat
Hans
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Kreipe
D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D019175:DNA Methylation / Q000235:genetics*; D006801:Humans; D035683:MicroRNAs / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods*; D013447:Sulfites / Q000737:chemistry
D035683:MicroRNAs; D013447:Sulfites; D004247:DNA; C042345:hydrogen sulfite