I am performing a ChIP-qPCR for a target promoter of interest. The binding site is on the negative strand upstream of the TSS. I'm using enzymatic digestion and see a nice digestion pattern. I am using IgG (-) control, and H3/polII as the (+) controls. I have positive primers where my TF of interest should be binding as well as a negative control ( GAPDH). My issue arises during the qPCR analysis. I am getting 'binding' of my TF of interest to the GAPDH site, i.e there is a fold enrichment compared to IgG. I am seeing fold enrichment for all of the genes of interest, except a few sites reported in the literature. Any insight would be greatly appreciated.
Dear Alison,
I'm not sure, if i fully understand your problem/question. You are concerned that you get a signal higher than IgG at the GAPDH promoter, right? But is it comparable to your target gene promoter enrichments or lower/higher - in other words, what are the fold increases over IgG at each site? Do you assess IgG signals at all the regions separately or do you normalize to one test target?
Best,
Kevin
The fact that you have a fold enrichment to your negative controlcompared to IgG is not worrisome IF the binding to this engative control region is much inferior to your positive control regions.
Is the fold enrichment that you are seeing in your test regions higher than to your negative control region? If so, then no worries.
Otherwise use more than one negative control region in your tests. If something goes wrong with your negative control and you only have one you may have to repeat your experience.
Hi Alison,
As long as fold enrichment in your TF pulldown much higher than fold enrichment in IgG, you're okay. There's no need to worry.
The problem with Negative regions in ChIP analysis is always a dirty arena as you will never know if a region is indeed a negative region. It is therefore suggested to do a panel of negative regions.
Hope this helps.
Good Luck!
Utkarsh
Be sure you are using the correct negative control. GAPDH is a housekeeping gene. What TF -antibody you are using for ChIP?
You should better calculate the relative fold enrichment in your interested promoter as compared to GAPDH promoter region.
While the importance of a negative control is beyond doubt in any foolproof experiment, its use in ChIP still demands deeper insight. Each ChIP study focusses on the binding of a unique TF to a unique site hence determining the perfect negative control is not realistically possible in most cases like here.
It would be nice if the scientific community took fresh initiatives in determining the exact need of this control and finding some better alternative.
Rightly said in an earlier post here the negative control is a dark arena in ChIP, yet we have to do it for the sake of data publishing pressures.
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