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Table 1 Restriction enzyme qPCR and dPCR

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

Expected methylation (%)

Average methylation ± standard deviation (%)

qPCR

dPCR

MDRE

MSRE

MDRE

MSRE

100

97 ± 1¥

100

94 ± 6*

100

90

87 ± 6+

85 ± 4

86 ± 6

63 ± 12

75

79 ± 8‡

69 ± 17

74 ± 13

93 ± 32

50

42 ± 22¥,+,‡

36 ± 11*,¥,+

59 ± 16*

55 ± 11*,¥,+

25

25 ± 9

21 ± 4*,‡

31 ± 17

24 ± 7*,‡

10

22 ± 20

7 ± 1¥

11 ± 28

12 ± 5¥

0

0

0+,‡

0

0+,‡

  1. The average % methylation calculated from three independent qPCR and dPCR measurements of a panel of methylated/unmethylated DNA standards using Methylation-Dependent Restriction Enzyme (MDRE) and Methylation-Sensitive Restriction Enzyme (MSRE) treatments; Each PCR measurement was performed using an independent RE digestion as template. Data normalised to the 100% methylated sample for MSRE assays and 0% for MDRE assays. Statistical comparisons using a One-Way ANOVA were performed on the data where each RE class was used within its optimal template range (0-50% for MSRE; 50-100% for MDRE); all comparisons between samples that were significant at the level of p < 0.05 are shown with pairs of the same symbols (*,¥,+,‡) denoting which two samples were compared, e.g. for MDRE dPCR, only the 50% and 100% methylated (expected % methylation) data points were significantly different from each other at the level of p < 0.05. Data points that were outside the viable range of the assay (< 0% or > 100% methylation) were removed from the analysis; all experimental conditions were n = 3 with the exception of the following that were n = 2: 10% and 25% MDRE qPCR and 90% MSRE qPCR (expected % methylation).