panomics promoter methylation PCR kit - results not what i expected (Sep/12/2008 )
I recently used the panomics promoter methylation PCR kit on gDNA from two different cell lines. Really quick you cut gDNA with MseI and bind MeCP2 to methylated regions and select for these methylated regions and perform PCR to amplify these regions. I know where the CpG island in my promoter is so was able to develop my own primers rather than using randoms.
So, theoretically, one cell line should have a methylated promoter and the other should be unmethylated. However, after the PCR I got a band for two segments (out of three) for the cell line that should be unmethylated while only getting a band for one segment (out of three) for the cell line that should be methylated. I have an idea the promoter is methylated in one and not in the other because of previous AZAD experiments, where we saw an increase in promoter activity in the methylated cell line and no change in the unmethylated cell line.
Any ideas how I got these bands that I shouldn't have? Faulty equipment, user error or am I wrong about my methylation hypothesis?
PCR is a very sensitive method of picking up minute quantities of template, so you may have saturated your reaction with the number of cycles. Or the MeCP2 enrichment didn't work properly or your digestion didn't work properly. You would need to work out if all these steps have worked efficiently.
The first port of call would be the PCR. I assume by your comments you are looking at an imprinted promoter? I think the kit maybe too crude to look at this.