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% Input or % methylation better for MeDIP-qPCR?


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#1 cnmiller



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Posted 08 June 2015 - 10:02 AM

Which one is more preferable? The kit that I am purchasing (Diagenode) appears to come with positive controls for 5hmC, 5mC, and an umethylated sample. Thus, I can calculate % methylation. However, I see that in the literature calculating your data to % input is much more prevalent.


So which way should I go?

#2 methylnick



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Posted 08 June 2015 - 07:51 PM

there is no "gold-standard" for this. Each has it's own underlying assumptions. Calculating by %methylation using the unmethylated control assumes that the unmethylated control is truly unmethylated. Further, that the genomic complement of your controls are equivalent to the samples you are testing (eg: they are genomically balanced), if you have CNV in your samples that deviate from your controls there is potential of mistaking DNA methylation with copy number aberration.


Going with %input is more prevalent because it inherently takes this genomic balance/imbalance into account. However, you are then making the assumption that any enrichment you are seeing over and above your input is in fact methylation and that your antibody is actually binding to, and detecting methylated DNA. 


Plenty of literature around both issues and concepts.

Good luck!


All comments and communication are my own personal ones, and are not tied to any of my affiliations. 

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