help with ChIP control--urgent! - (Oct/24/2005 )
ciao a tutti..
well.. I'm looking for any kind of information about controlls of Chip, that is..
which are the cnotrolls of the PCR of a ChIP? Do I have to put in comparison the intensity of the sample with the input or with the internal controll(actin)?
thanks a lot!
marghe
If you expect your gene to be actively expressed by hyperacetylation then you can use actin as control. But if the gene of your interest is silenced by HADC, because absense of signal here indicates deacetylation, I don't know what control you should use.
well.. I'm looking for any kind of information about controlls of Chip, that is..
which are the cnotrolls of the PCR of a ChIP? Do I have to put in comparison the intensity of the sample with the input or with the internal controll(actin)?
thanks a lot!
marghe
You can use Input or pre-IP supernatant as your control since you know what is your gene expression (antibody treated). Therefore, Input could be positive or negative control.
Yes, input DNA is used as control for quantity of starting DNA. But DNA may be lost during subsequent washs and precipitation, how can we control that? For example, I am precipitating DNA using anti-H3K9 methylation and DNA from my treated cells is lost during washes and as a result, those samples don't give a signal while untreated cells do. This result may be mistakenly explained as the result of H3K9 methylation loss. There is no control for this. The only thing I can do is to repeat the experiment again and again to see if it is reproducible. Anyone has good idea?
This is exactly my problem!! I don't know.. In the laboratory where I worked this summer they calculated the ratio between the sample the input and the signal from actin.. but I'don't.. What I thought is to calculate tha ratio only between the signal of the sample and the actin of the same ChIPs.. 
pcrman,
a good way to see if your are not losing your DNA through washes is to try a CHiP-PCR with something that is known to associate with your protein of interest.
For the case of H3K9 methylation, I know that there is a strong association with pericentromeric regions and you would get enrichment of them with an IP.
centromeric and pericentromeric regions are sinks for heterchromatin modified histones and hterechromatin proteins and can be used as positive IP controls in experiments.
Good luck
Nick
I've got lots of issues with the ChIP assay and the only way around this one (loss of DNA through the wash steps) is to do lots of replicates until you're really confident what ur seeing is real.