I'm trying to establish a method to detect methylation in the NR3C1 gene but I detect an additional peak in the melt profile. I thought about primer dimers, but I'm not sure that is it the problem. Indeed, the "high" of the additional peak is proportional to the real peak (which depend on how much methylated the sequence is).
I attach the file with the melt curves.
The difference plot also doesn't look very nice (attached).
One of the primer is ACCTAATCTCTCTAAAACGACGTTAA. I think it could be prone to hairpins or self hybridization.? Could I slightly modify it?
Thank you very much for any possible advise!