Determing if ChIP Signal Is Real
Posted 26 January 2010 - 09:33 PM
There appears to be quite a bit of variability in the literature in terms of how ChIP data is presented; from %input to specific/mock to just reporting the comparison of a control group to an experimental group. Thus I (and I imagine others) struggle to understand what a "real" ChIP signal is.
From an initial reading of the literature it seemed that specific/mock (e.g, IgG) would be a good way to determine if what one is seeing is real. However, the primary drawback I see to this is how to deal with highly variable mock data (At least that's what I get, sometimes it doesn't even show up after 40 cycles in some replicates, which makes it difficult to quantify (score it 40? throw it out?...)). However, in the only two published articles I've found that actually show their raw qPCR data the Mock data looks fairly clean and quite high! (See Hao, Liu, Gonye and Schwaber (2008) J Neurosci Meth 172, 38-42; and Nelson JD, Flanagin S, Kawata Y, Denisenko O, Bomsztyk K (2008) Am J Renal Physio 294, F525-33 in supplemental materials).
Is the Mock data in these articles (Cts around 25-30) what Mock data should look like? Or should it be difficult to get any signal?
The other suggestion that has been mentioned on this board is to use primers towards a genetic region that is not transcribed or does not bind the protein of interest. This approach makes a lot of sense to me, however it remains a bit more difficult to do than it seems on the surface. For example, I'm working with brain tissue (the hippocampus) so I figure using primers for rhodopsin as well as those aimed at clusters of olfactory receptors should be a safe bet in terms of genetic regions that should be shut down in an area that does not directly detect light or odors. However, upon doing ChIP for RNAPII (using 2ug of DNA, incubating overnight using Magna ChIP kit from Millipore) I find that I get a %input of these regions that is on par with my gene of interest as well as other genes that I know are transcribed in this brain region! However, the fold change over my mock IgG sample is low for rhodopsin and olfactory receptors (typically < 10 whereas in genes known to be transcribed it hovers around 20-60). BUT of course there is significant variability in the Mock IgG samples, so can this data be trusted? If %input values are similar but specific/mock is consistently different, does this mean these are good negative control regions? If not, anyone have a good suggestion for a negative control region? I'm thinking perhaps one solution is to find a way to increase my qPCR signal and thereby get cleaner Mock data like in the publications mentioned above? Perhaps by adapting the DNA purification of the FastChIP protocol?
Well I guess I'll leave it at that for now. Thanks for reading this far! Any insight is appreciated.
Posted 31 January 2010 - 04:14 PM
Posted 11 February 2010 - 12:57 PM
If you perform PCR on a negative control region and see a significant difference in % input then I think you are good to go. Like you I too saw a weak signal for Pol II at a gene that should not be expressed in my tissue of interest. However since my positive control region gave a stronger signal, I was fairly happy with it. I have also redesigned the primers a bit to move them to a more 3' intronic region (they were previously in an exon!). Regardless of the signal, intuitively this is a much better control and is a lot more rigorous......which is why I am sure many will turn away from it.