Hi,
I am trying to measure the differential methylation of the CpGs in a promoter region of my gene of interest. I have done bisulfite conversion on human genomic DNA extracted from blood followed by Nested PCR for at the region of interest and is able to obtain single band for my PCR. I did ExoSAP clean up on the PCR product followed by BigDye cycle sequencing on the ABI3130. I calculate the percentage of methylation at the CpGs by measuring the C and T peak heights at these CpG sites.
However, I am observing quite a bit of baseline noise in my electropherogram (~5-10% of the mojor peaks). Due to this noise, I am unable to be certain on the completness of my bisulfite conversion. Does any one experience the same problem? Any suggestions on how to improve this?
At the same time, I am getting inconsistent result when sequenced with forward and reverse primers. The percentage methylation observed on both the forward and reverse strand may differ by ~10-15%. Does anyone know how I can improve on this?
I would appreciate all your suggestions. Hope to hear from you guys.
Thanks.