5-Lipoxygenase DNA Methylation and mRNA Content in the Brain and Heart of Young and Old Mice
Figure 2
An example of the SYBR Green RD-qPCR assay of 5-LOX DNA methylation (shown are graphs obtained from 10 cerebellar samples). The samples were digested with the methylation-sensitive endonucleases (AciI, BstUI, HinP1I, and HpaII) as described in Material and Methods. The PCR reaction for the promoter-UTR (10 samples each: AciI = blue, BstUI = red, HinP1I = green, and HpaII = gray) and corresponding input control regions (shown in yellow) were carried out in separate tubes. Panel A shows the dissociation curve data, which indicate the presence of only one PCR product (peak) for each specific set of primers (fluorescence (first derivative of the raw fluorescence reading multiplied by ) on the Y-axis versus the PCR product melting temperature (°C) on the X-axis). Panel B shows examples of the amplification plots used for calculating the quantitative data (the amplification plots fluorescence (baseline-corrected raw fluorescence) on the Y-axis versus cycle number on the X-axis). In this assay, the threshold cycle is inversely proportional to the log of the initial copy number. In other words, the more template that is present initially, the fewer the number of cycles required for the fluorescence signal to be detectable above background.
