Hi,

what is the soruce of your control DNA for methylated/ Unmenthylated sequence? By nature most methylated control DNAs are generated by using mehtyltransferases like SSS which might not be 100% effective, so this might be the reason for getting values above 100% for your samples.

In case you know sample types or cell lines with 100%/0% methylation you could mix the DNAs to generate the standards, but make sure, that both samples types do contain the same amount of amplifyable DNA after your BS-conversion (e.g. run them through qPCR and check if both samples give you the same Cq value and then you could mix them based on these results)

Sven

I agree with Sven Reister that adding more standards than just the absolute 0% and 100% makes reading of the curve more accurate.

You can also run a methylation-specific ALU PCR after MsSSI treatment & bisulfite conversion, to make sure the DNA is methylated.

Hello Sven Reister and Zahra Haider

The source of my controls are : According to the kit inser- in vitro methylation of the control DNA was achieved using SssI methylase. Bisulfite conversion of control DNA was achieved using the EpiTect Bisulfite Kit.

EpiTect PCR Control DNA Set- Human control DNA set (containing both bisulfite converted methylated and unmethylated DNA and unconverted

unmethylated DNA)

I do mix 0 & 100 in equimolar conc to get the various levels, like 0%, 25%, 50% & 100%.

However, after several trials also, a few samples are showing the graph to be above 100%