The goal of the PLOS ONE study by Leontiou et al. was to compare the conversion efficiency, DNA degradation and conversion specificity of four widely cited kits for bisulfite conversion, including the MethylEdge™ Bisulfite Conversion Kit. To assess the ability of each kit to detect methylation, a mixture of methylated and unmethylated λ-DNA fragments were used as the test substrate in varying ratios of methylated to unmethylated DNA for use in two downstream applications: PCR and next-generation sequencing. Initial estimates of conversion efficiency were assessed by PCR amplification of bisulfite converted DNA using primers specific to unmethylated sequences; no DNA was amplified under these conditions. However, when sequencing on an Illumina MiSeq platform, differences among the kits became more obvious. The MethylEdge™ Bisulfite Conversion Kit had the highest conversion efficiency (99.8%); the lowest conversion efficiency among the kits was 97.9%. DNA yield and degradation after bisulfite conversion were determined using the ratio of DNA measured by fluorescent DNA-binding dye quantitation of 12 samples before and after conversion. The highest DNA yield was an average of 55% for a competitor kit; the MethylEdge™ Bisulfite Conversion Kit averaged 52%. The kit with the lowest DNA yield came in significantly lower at 33%. Conversion specificity, that is the conversion of unmethylated cytosines to uracils, was determined by adding methylated λ DNA into unmethylated λ DNA at specific percentages (e.g., 0%, 25%, 50% and 100% methylated DNA) and sequencing after bisulfite conversion. The expected methylation and observed methylation of the 26 CpG sites in the λ DNA were plotted to determine the correlation coefficient; the MethylEdge™ Bisulfite Conversion Kit and one of the competitor kits had the strongest correlation compared to the other tested kits. Based on these results, the MethylEdge™ Bisulfite Conversion Kit and one other kit were ranked as the best performers and further analyzed for bisulfite conversion specificity by sequencing the conversion products on an Illumina MiSeq platform. Again, standard curves were generated for both conversion kits to assess the correlation of the expected methylation and observed methylation of the CpG sites across the λ DNA. The value for the MethylEdge™ Bisulfite Conversion Kit (r2 competitor kit (r2 = 0.99) was just above the = 0.97). Because researchers were looking for a bisulfite conversion kit for which “…the desired outcomes (conversion of unmethylated cytosines) positively correlate with the undesired side effects (DNA degradation and inappropriate conversion)…”, the MethylEdge™ Bisulfite Conversion System, with the highest correlation coefficient of the four kits tested and most accurate quantitation of cytosine methylation, was the system the authors selected for further use in their methylation studies. Learn More about MethylEdge™ » Read the PLOS ONE Article » enews | Spring 2016 Pagina 16
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