I'm doing ChIP assays, and I'm not sure how to perform the qPCR. Maybe it is a stupid question, but I prefer to ask. I measured the DNA after the ChIP assay and I had a different concentration in each sample, between the control and the treatments in AcH3 for example. I took the samples and I did the qPCR using the DNA from the ChIP, doing the same dilutions for each sample. When I saw the qPCR I was wondering if the difference that I saw in the results were real or they just were due to the different concentration of DNA, for this reason, I did another qPCR, but this time I add the same amount of DNA, and the results were more or less the same than before. So now I don't know how I should do it. Can someone help me? Thanks in advance
For ChIP, each sample should first be normalized against its own input. So this will take care for the possible difference in DNA amount prior to doing the immunoprecipitation. Before you do the IP, you need to set aside a fraction of sample for input. Then after the ChIP, you get your IPed DNA. After reverse crosslinking both the input and eluent samples, then qPCR will be performed on these DNA. There is really no need to dilute your DNA samples at this point. Based on the raw Ct for your input and eluent, you can calculate fold enrichment for your ChIP.
Here is an example of how to calculate the raw Cts:
https://www.lifetechnologies.com/us/en/home/life-science/epigenetics-noncoding-rna-research/chromatin-remodeling/chromatin-immunoprecipitation-chip/chip-analysis.html
Ok! Thanks Augusta!
Of course I add the same DNA amount when I start the ChIP assay, I mean, after sonication I use the same concentration of DNA in the immunoprecipitation.
Thanks!!!
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