Clin Epigenetics ; 12 1 : 90, 06 Therefore, there is a growing interest in assessing the clinical utility of DNA methylation as a biomarker for diagnosing disease and guiding treatment. The development of an accurate loci-specific methylation assay, suitable for use on low-input clinical material, is crucial for advancing DNA methylation biomarkers into a clinical setting.
A targeted multiplex bisulphite PCR sequencing approach meets these needs by allowing multiple DNA methylated regions to be interrogated simultaneously in one experiment on limited clinical material.
We describe additional steps to improve performance and reliability: 1 pre-sequencing PCR optimisation which includes prostate cancer enzyme marker the optimal PCR cycling temperature and primer concentration and 2 post-sequencing PCR optimisation to achieve uniform coverage of each amplicon.
We use a gradient of methylated controls to demonstrate how PCR bias can be assessed and corrected. Methylated controls also allow assessment of the sensitivity of methylation detection for each amplicon.
Here, we show that the MBPS assay can amplify as little as 0.
Furthermore, the multiplex bisulphite PCR assay can comprehensively interrogate multiple regions on ng of formalin-fixed paraffin-embedded DNA or circulating cell-free DNA. The optimisation and additional quality control steps described here improve the performance and reliability of this method, advancing it towards potential clinical applications in biomarker studies.