Figure 7.

The use of bisulfite-qPCR to detect methylated/hydroxymethylated DNA and loss of methylation after decitabine treatment. (a) Validation of bisulfite-qPCR for Oxytricha trifallax DNA. The ddCt method was used to quantify the loss of signal induced by bisulfite treatment relative to signal from an equal amount of untreated DNA. Signal was normalized to total DNA used as input for the bisulfite treatment. A PCR fragment of Contig4414 amplified from vegetative (nonmethylated) DNA provided the conversion control (negative control for methylation); two indepdendent bisulfite treatments of this PCR product, A & B, were used as templates in qPCR. All qPCR was performed in triplicate and the average is plotted with standard error. The Student's 1-tailed t-test for unequal variance was used and p-values are indicated: *, p < 0.05; **, p < 0.01, ***, p < 0.005. Values marked *** in Figures 7 and 8 appear significant even with a correction for multiple tests. (b) Staging data for 40 h decitabine (dAza)-treated cells. Cells were fixed and DAPI stained to allow staging based on nuclear morphology as in Figure 2e. (c) Bisulfite-qPCR analysis of decitabine-induced demethylation in 40 h cells. Both native (red bars) and bisulfite-converted DNA (green bars) are shown, normalized to native mitochondrial rDNA signal (for loading) and to untreated cells (grey bars) to determine fold change. All qPCR was performed in triplicate and the average is plotted with standard error. Statistical test for significance was carried out with Student's 1-tailed t-test (*, p < 0.05; **, p < 0.01, ***, p < 0.005). DAPI, 4',6-diamidino-2-phenylindole; qPCR, quantitative PCR; Veg, vegetative.