Relative quantitation of DNA methyltransferase mRNA by real-time RT-PCR assay.

Abstract

DNA methylation is one mechanism of epigenetic gene regulation and influences gene expression by recruiting methylcytosine-binding proteins and/or inducing changes in chromatin structure. In mammals, DNA methylation is mediated by at least four DNA methyltransferase (Dnmt) enzymes, including Dnmt1, Dnmt2, Dnmt3a, and Dnmt3b. To understand fully how DNA methylation is involved in gene regulation, knowledge of Dnmt mRNA transcript levels is required, both as a surrogate measure of Dnmt protein levels and also to facilitate an understanding of the regulation of expression of the corresponding genes. Measurement of transcript levels has traditionally been achieved by Northern blot analysis and more recently either by the ribonuclease protection assay or by reverse-transcription polymerase chain reaction (RT-PCR), followed by agarose gel electrophoresis. In the past few years, a form of PCR has been developed that measures the accumulation of PCR product in real time. In conjunction with RT, real-time RT-PCR has become a widely accepted tool for measuring mRNA transcript levels and is now probably the method of choice. This technique is both sensitive and specific and allows for the rapid assessment of Dnmt mRNA transcript levels as well transcripts for other genes that may be involved in DNA methylation.