Problems with the study of promoters methylation - (Jan/08/2016 )

I would like to examine whether the promoters of isoform A and B of the progesterone receptor are subject to methylation in the corpus luteum during the estrous cycle in cows. I designed the primers for each isoform to methyl specific PCR. Methylated and unmethylated specific primers were designed with using MethPrimer web page. I performed PCR using Hot Start Page polymerase and bisulfite converted genomic DNA as template. The obtained results were demonstrated partial methylation of both isoforms. Both pair of the primers for methylated and unmethylated sequence showed bands on agarose gel. The assessment of the degree of brightness of the bands on the agarose gel were suggested not 50% methylation. I wanted to confirm these results by sequencing the fragments of the promoters of both isoforms of DNA after conversion and compare to the reference DNA to see what percentage of DNA is methylated in both promoters of A and B isoforms. However, sequencing revealed that the both promoters are not methylated (100 % unmethylated). Is this situation is at all possible. I'm trying to find a solution and nothing comes to mind.

Hi Robert, sorry for the hiatus. 

Do you have known controls to ensure your primers sets actually work? It is possible to have heterogeneous methylation. 

Nick