Table 2.

Comparison of Quantitative MSP and MIP Assays for Quantitative Assessment of CpG Methylation

	Methylation-specific quantitative PCR (MethyLight, etc.)	Methylation-independent PCR (for subsequent pyrosequencing, mass spectrometry, COBRA, etc.)
Basic characteristics	Quantification during PCR	Quantification after PCR; not specific for the methylated or unmethylated sequences
Paraffin-embedded tissue	Usable	Usable
Precision	Good	Good, especially at high-level methylation
Accuracy	Good	Good, especially at high-level methylation
Monitoring of complete bisulfite conversion	By amplification of a non-CpG genomic reference*	By the presence of non-CpG cytosine in templates that should be completely converted
Genomic reference to measure the amount of input bisulfite-converted DNA	Necessary*	Unnecessary (measuring both methylated and unmethylated sequences)
Resolution	Lower; block of CpG sites coincident with primer and/or probe sequences	Very high (single nucleotide level)
Analytical sensitivity	Very high (1% methylated sequence)	2% to10% methylated sequence (depending on subsequent detection method)
PCR design	Easy for high density CpG sites (applicable to most CpG islands)	Easy when there is a small CpG island abutted by CpG sparse areas. CpG sites within PCR primers must be limited
Closed system versus opening of PCR tubes	PCR tubes always closed	Opening of PCR tubes usually necessary
Samples for standard curves	Necessary	Unnecessary
PCR bias	Specific for methylated sequence by definition	Need to minimize PCR bias between methylated and unmethylated sequences

^*Some variants of real-time PCR assays do not require a genomic reference.