Quantification of epigenetic biomarkers: an evaluation of established and emerging methods for DNA methylation analysis

Table 2 Methylight qPCR and dPCR

Expected methylation (%)	Average methylation ± standard deviation (%)
	qPCR	dPCR
	P14_M	P14_M	P14_M	P14_M2	P14_M + P14_M2
	Singleplex	Singleplex	Duplex	Duplex	Duplex
100	100^*	100	100	100	100
90	78 ± 4^¥	77 ± 27	99 ± 19^*	86 ± 20	92 ± 19
75	66 ± 18^*	81 ± 13	86 ± 20	77 ± 17	81 ± 19
50	49 ± 10^¥,+	52 ± 16	58 ± 10^*,¥	57 ± 28^*	56 ± 18^*
25	28 ± 4^‡	21 ± 7	31 ± 5	28 ± 2	29 ± 4
10	13 ± 3⁺	17 ± 4	18 ± 6^¥	9 ± 15^*	13 ± 10^*
0	0^‡	0	0	0	0

The average % methylation calculated from three independent qPCR and dPCR measurements of three independent bisulfite conversions of a panel of methylated/unmethylated DNA standards; Data for dPCR show measurements from the P14_M assay used in singleplex and for the P14_M and P14_M2 assays used together in duplex, showing the % methylation for each assay when analysed individually and with the estimated targets combined (P14_M + P14_M2). Symbols denote statistical comparisons using a One-Way ANOVA test. All comparisons between samples with expected differences in methylation of ≥ 50% were significant at the level of p < 0.05 (with the exception of 50% vs. 100% methylation with MethyLight P14_M2 Duplex) and are not shown; all comparisons between samples of differences between 0-40% expected methylation that are significant (p < 0.05) are shown with pairs of the same symbols (*^,¥,+,‡) denoting which two samples were compared, e.g. for dPCR P14_M2 duplex, only the 10% and 50% methylated (expected % methylation) data points were significantly different from each other at the level of p < 0.05.

ISSN: 1471-2164