Hello, everyone, I am currently investigating the methylation status for a gene of interest by pyrosequencing, but the result is not reproducible.
Basically, I extract the DNA from a certain cell line, then perform the bisulfite treatment. afterward, I make it triplicate for one sample in PCR and pyroseq.
The strange thing is the methylation percentage for the same CpG site is different in the triplicate, such as 1%, 2%, 50% in triplicate, or 50%, 30%, 20% in triplicate.
the Results of the same sample are also different between the different independent pyroseq assay...
I am really confused about the result. does it mean my DNA sample is a mixture or any other reason?
But the DNA samples should be mixed up thoroughly, even though it is a mixture....
There are several potential causes of non-reproducibility in the study of methylation rates using pyrosequencing:
Overall, it is important to carefully consider all potential sources of variability when conducting a pyrosequencing study of methylation rates, and to take steps to minimize these sources of variability in order to obtain reproducible results.
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