Global DNA methylation measurement by HPLC using low amounts of DNA

Abstract

Epigenetic changes in chromatin structure can influence gene expression without affecting the DNA sequence. The most commonly studied epigenetic modification, DNA methylation, has been implicated in normal tissue development and disease progression, and can be influenced by diet and other environmental factors. Current HPLC methods of determining DNA methylation may require relatively large amounts of DNA (50 μg); as many tissues have low DNA yields, this can be hard to achieve. We isolated DNA from mouse colon and liver in a study investigating post-natal supplementation with selenium and folic acid. After optimizing the methods to account for lower initial DNA amounts, we digested 3 μg of DNA to deoxynucleotide monophosphates, then purified and quantified it. Samples were analyzed by reversed-phase HPLC to determine global DNA methylation levels using commercial nucleotide standards. The HPLC column was cooled to 6(C (reducing run time), and detection was at 280 nm (UV). We showed that methylated cytosine can be accurately and reproducibly measured in as little as 3 μg of DNA using this HPLC analysis method (within-assay CV <2%). We also used this method to detect reduced DNA methylation in liver (P = 0.009) in response to post-natal supplementation with selenium and folate.