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In the range of currently used methodologies in methylation studies (reviewed in Reference 1), sequencing of bisulfite-treated DNA (2) can be considered the gold standard, as it reveals the methylation status of each CpG dinuclotide. Nevertheless, this technique is relatively expensive, therefore it has rarely been used in large-scale experiments. By contrast, the simplicity of design and easy performance of methylation-specific PCR (MSP) (3) has made it the most widely used technique in the investigation of methylation status of the genes. However, MSP is prone to false positive results and may lead to an overestimation of the number of methylated samples (4).

Reversal of PCR bias for improved sensitivity of the DNA methylation melting curve assay