Hi. I am doing chip-qpcr and have analyzed the region using specific primers for the GOI. The sonication has given me sheared DNA in the range of 700 bp to 2.5 kb size. I have 4 different regions (such as hormone response element site, SP-1 site and NF-KB binding site etc) that are located within 500bp of transcription start site of the GOI. The problem is I found my protein to be binding in all of these regions as the sonication yielded 2.5kb to 700bp. Further the fold enrichment was 100fold compared to an IgG control. Moreover, if I generate chromatin fragment of 200-1000 bp range, how can I differentiate one region vs. the other as each of them are 100bp apart only. For example if the sonication has resulted in 500bp chromatin size and after antibody pull-down and qpcr, all my primers (scanning hormone response element site, SP-1 site and NF-KB binding site etc) will generate amplicons.
Also I was wondering if I could re-sonicate the purified DNA (DNA eluted after the reverse cross-linking of Protein/DNA Complexes and column purified) ?
Can I do restriction endonuclease digestion of this DNA and do qPCR (if I find it to specifically separate the 100bp regions)?
Thank you all in advance.
Kejun Guo. Thanks for the suggestion. I agree with what you are saying but I just need to prove that my transcription factor does bind to the promoter region of my GOI (which I see that it does). The only thing is I can't pin point as to where exactly it binds as it is known to heterodimerize with the other transcription factors like SP-1 and NF-KB.
I agree with Kejun. To answer your second and third question, I don't think re-cutting the DNA after elution will yield the results you are looking for, if I understand your question correctly. If you think about it, the protein has already pulled down the DNA with it, so even if the additional sonication separates your regions of interest, that doesn't mean they were differentially bound to the protein or bound the same or any of that. The whole 2kb of DNA has already immunoprecipitated; fragmenting it further doesn't change what came down, it might just bias your results with more stable DNA templates or intact amplicons vs those that have been disrupted by the cutting.
As a side note that doesn't directly relate to this question: if possible, I would try to get a better sonication efficiency for future ChIP experiments. These fragments are far too large to produce reliable results, you should be aiming for 250-500bp fragments.
Thanks Jim Rosa. Yes that is true. I have already pulled the DNA down and therefore it will amplify the regions even if I cut it further. However is there a way that I can produce 200bp fragments and not more. Because as I said the sites are 100bp of each other.
I don't think ChIP-qPCR will be able to differentiate between sites that are 100bp apart from each other. You could try to get very small fragment sizes (
Thanks Jim Ropa. I agree that sonication is the most crucial step which needs to be optimized for the current cell line (myometrial cells) that I am working with.
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