Methylation of cytosine at gene promoter regions may be diagnostic of cancer. However, there are many stages in the measurement of DNA methylation where inaccuracies may occur and this may prevent the use of these measurements in the clinical setting. The International Bureau of Weights and Measures (BIPM) has sponsored a project that aims to improve the accuracy of quantitative DNA methylation measurements. The first stage of the project involved preparation and extensive characterisation of two partially methylated 100 base pair (bp) DNA amplicons at the Korea Research Institute of Standards and Science (KRISS). The National Measurement Institute of Australia (NMIA) developed a liquid chromatography-mass spectrometry (LC-MS) method for measuring the degree of methylation in these samples and the values obtained agreed closely with the reference values assigned. This paper describes how development and validation of the LC-MS measuring system enabled metrological traceability to the SI mole and how the study results provided evidence that there may be a new mechanism for calibration of DNA methylation using sub-microgram amounts of reference material. This is a significant outcome as it raises the possibility that reference materials with known methylation ratios that are traceable to the SI could be made for important genes and these could help evaluate the clinical utility of diagnostic and prognostic gene methylation measurements.
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