This study, while relying on the less-precise 5mC immunoprecipitation method for detecting DMRs, took advantage of a medium-throughput validation technique. Among the examples covered here, this was the first study to attempt the validation of a substantial set of DMRs. Unfortunately, the mass spectrometry-based method, while highly sensitive to measuring methylation proportions in each CpG, is too expensive for most users and too difficult to scale up for a more complete validation series. Furthermore, the proportion of genes validated (12 out of 26) seems relatively small. This could indicate that the immunoprecipitation/microarray screening produced many false positives or that the validation method was not sufficiently sensitive to detect small changes in methylation. Since methyl immunoprecipitation methods are by their nature less precise than bisulfite methods, we assume that screening was the problem in this analysis. Alternatively, the 1.3-fold cut-off may have been too close to background noise levels to reliably select candidates. No matter which explanation is true, the discordance between screening and validation shows that a recursive consideration of screening methods could have improved the choice of filtering methods.
