Control DNA in bisulfite methylation analysis - (Jan/16/2006 )
Hi, I am a newer in DNA methylation field. I want to make sure about the positive control and negative control. Does the positive control means that I should mathylate my own sample DNA using Sss-1 first and then treat it with bisulphite? How about the negative control? Does it need to be demethylated the DNA sample? Could you give me some advices? Thank you!
Most people use peripheral blood cells (PBC) DNA as negative control, because most genes are supposed to be unmethylated in PBC compared to cancer. Although demethylation of DNA can be an alternative, the existence of an active DNA demethylase has not been confirmed yet.
Thank you very much!
Thanks a bunch!
I am still confused wether I should use Sss1 to treat my genomic DNA as a positive control or to treat a plasmid containing the gene sequence before bisulphite treatment? Can I get the positive control from the company?
The peripheral blood cells as negative control include erythrocyte, leukocyte, and platelet, Which is the best part I should take?
Thank you again!
Both genomic DNA and plasmid can be used to generate methylated control DNA which is then bisulfite modified as you do with your study samples.
Chemicon sells such control DNA. If you want to generate your own control DNA using Sss I methylase, read more on NEB site about this enzyme.
For negative control, I mean Peripheral blood mononuclear cells (PMBC). Sorry.
Do you have a reference for this?