Abstract

Using High Throughput qPCR for DNA Methylation Biomarker Validation

Andreas Weinhaeusel, Senior Scientist, Austrian Institute of Technology GmbH

Elucidation of genome wide epigenetic changes has become a routine application, and is of utmost interest for the development of biomarkers. Along the biomarker developmental chain upon discovery of candidate markers, confirmation and validation are indispensable steps. For nucleic acids based markers the method of choice is qPCR. Focusing on DNA methylation based biomarker development, we have established methylation sensitive restriction enzyme (MSRE) based qPCR analyses for DNA methylation analyses. Efficient assay design tools have been developed enabling high throughput sequence manipulation and qPCR design. Several hundred assays for human DNA methylation targets have been designed and setup. Analytical validation according MIQE guidelines has been conducted using a standard qPCR and Fluidigm’s Biomark system. These assays have been used for validation of DNA-methylation biomarkers of cancerous and non cancerous disease. Results from the high throughput MSRE-qPCR Biomark runs were successfully used for confirmation of DNA methylation changes elucidated by Illumina’s bisulfite deamination based 450k genomic methylation arrays. From our experience the MSRE high throughput qPCR enables reliable and very efficient validation of DNA methylation changes derived from genome-wide analyses.