ChIP on chip - simple questions (Aug/04/2008 )

Hey guys,

I am new to ChIP technology. Thus I have some simple aka stupid questions regarding on ChIP techniques.

1) How can I determine the difference in the results? How much fold change is considered as significant difference? (or is it depends on software as well as companies?)
2) What are the other ways to validate microarray results other than real time PCR?

thanks! looking forward to hearing responses....

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Difference in the results: we determine the amount of pulled down Chromatin (for one primer set) as % of input (typically I can get say 10^-5 for a negative region vs. 10^-3 for a positive region. The important part is to not only compare the positive region to a restult from an isotype control antibody but also compare a positive region against a definitively negative region (e.g. an ORF)and to calculate the fold change from there.
significance: depends on the antibody you're using. 2fold can be significant. you have to do some statistics.



to look at binding...nothing that is simpler. more difficult, but probably possible are EMSAs. and to look at the functional level: clone your enhancer region of interest in front of a luciferase gene, cotransfect with the transcription factor, the read-out is luciferase activity.
but ChIP-qPCR is by far the easiest.

have fun

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