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We have recently been trying to establish a working MeDIP protocol using a commercial kit.
We have implemented a quality control step before putting our DNA through amplifcation/fluorescently labelling stages and onto chips, to ensure the MeDIP is working efficiently
We take even amounts of Reference/Input DNA and IP DNA and run these together in a qPCR for Beta-actin (as an unmethylated control) and H19 + TCE (which should be imprinted/methylated).
We are running into problems;
- We get SOME beta-actin amplifying in our MeDIP fraction (admittedly it is far less than the reference, but it is still there)
- We are seeing more amplification (earlier amplification, lower ct values) in our supposed methylated target genes in the INPUT/REFERENCE samples than the MeDIP fractions, even though the same amount of DNA is loaded per PCR.
Can anyone suggest explanations or relate experiences on this matter? We are also trying to get our heads around the concept of precisely how much DNA we should be pulling down from the IP ie. Should the MeDIP fraction contain/represent many many fold times the difference of methylated CpGs than the reference sample?
Additionally, does anyone have any experiences related to MeDIPs that feature;
a ) No denaturation stage
b ) No proteinase-K elution stage
Thanks!
