I am performing methyl-specific PCR to determine the level of DNA methylation in the promoters of the isoforms A (PGRA) and B (PGR) of the bovine nuclear progesterone receptor gene. For each isoform, two pairs of primers and probes were designed for methylated (M) sequence and unmethylated (U) sequence. The primers were designed in Methyl Primer Express. However, a problem arose that I can't interpret. The agarose gel image shows that the PGRA isoform is more methylated than the PGRB isoform. When reading the Ct in real-time PCR for PGRA, there should be a smaller Ct for M primers (there is more methylated product), while a higher Ct should be for U primers (there is less unmethylated product). In the case of PGRB, the Ct values for M and U primers should be the opposite as for PGRA primers (less methylated and more unmethylated product). This would indicate less DNA methylation of PGRB. The Ct values read for the example M-PGRA (Ct 36.2), U-PGRA (Ct 30.1) and M-PGRB (Ct 34.4), U-PGRB (Ct 28.4). Unfortunately, the Ct values for PGRA and PGRB do not match to results what I obtained on the agarose gel. Is it at all possible to get a different result on an agarose gel and a different result from a real-time PCR reading? Could any of you help me interpret these results?