controls for chip-seq real time PCR of h4k16Ac (293 cells)
Hey everyone,
I'm preforming a Chip-seq experiment on H4K16Ac using HEK293 cells. I would like to have both positive and negative controls for real time PCR verification.
Does anyone have had experience with this protocol and can share the controls used?
Thank you
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The problem with ChIP-qPCR is that there is not really a way to robustly assess what is a positive- and negative region without having ChIP-seq itself. Different genomic regions can have different sorts of amplification bias, so negative regions (that do not have the protein) could just amplify well in PCR, due to GC or other biases, skewing results. You need several negative regions to get a good estimate what the background is. Positive controls should be based on your data or the literature. But frankly, I would not bother with qPCR unless an anachronistic reviewer asks for it. Just do a good experimental design on your ChIP-seq, with good replication and analysis and call it a day.