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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).