what is meaning of true enrichment for chip qPCR? - (Oct/19/2007 )
Dear All,
I am very confused and desparated to interprete ChIP qPCR data enrichment. Pls help.
If I use SAME amount (5ng) of input DNA and IPed (e.g acetylated H4) DNA for qPCR, is that true that the IPed signal should be higher than the input signal if my target gene is an active gene and highly acetylated? What happen if the IPed signal is same to input signal (same Ct count), while IPed signal is much higher than negative control, normal rabbit IgG (also used 5ng for qPCR)? is there enrichment or not? What's meaning of enrichment? Thank you so much.
hk
Enrichment doesn't mean the comparison between IPed and input, or IPed and negative controls. Enrichment only make sense when you compare samples of different treatments, different cells, different conditions. for example, one gene expresses in one cell but not in another cell, you use the two cells to do Chip, you find signal for h3 acetylation is higher (enriched) in the expressing cells than the non-expressing cells, then you can say that h3 acetylation has something to do with the gene's expression.
I know this is an old post, but I'm going to bring it back to life because I've recently had this question come up and want to clarify for myself and others.
There are a lot of posts on this (fantastic) forum about ChIP, qPCR, Ct counts, etc and how to measure and qualify a good IP. As pcrman stated above, the real point of ChIP-Chip is to evaluate changes among different cells under different conditions, etc. HOWEVER, this doesn't apply to the first question most ChIPpers come across: Did I do a good IP? How do I compare my positive IP versus my negative? If you use qPCR, and use the same VOLUME of pos. and neg. IP solution (regardless of DNA concentration), then the results will be dependent on the way you ran your assay - did you handle the positive and negative samples EXACTLY the same? BUT, if you add the same AMOUNT OF DNA, is this a fair comparison of the two samples (i.e. if your positive IP is at 40 ng/ul, and your negative is at 10-15 ng/ul, is 20ng of each sample a good comparison)?
I believe that (and by all means would love to hear input from others out there) that qPCR should be done with equal VOLUMES of positive and negative IP product, with as much care as possible placed upon protocol cleanliness. I say this for two reasons: 1) an effective IP should naturally yield a lesser quantity of DNA for the negative IP than the positive, and 2) 'normalizing' the quantity of DNA for qPCR unfairly biases towards the background negative sample. The results of an equal amount of DNA in a qPCR has, at least by my experience, resulted in Ct values that are very close, but still show higher quantities of positive control (i.e. positive IP = 35-ish and negative=36-ish).
If anyone else has some insight into the 'art' that is the ChIP and ChIP-Chip, especially in regards to that fuzzy transitional area between IP and hybridization, I'd love to hear it.
CB
For our Chip-chip (histone acetylation) first we compare our IP sample with the IgG sample (as these have been treated exactly the same way). If we see enrichment at our positive control area but not at our negative region we go onto the next step...
After we amplify our DNA we do another round of qPCR. This time, we compare the Input to the IPed sample. Normally we find that the results are similiar to the IP vs IgG qPCR.
That's about it really. It seems to be working fine and we're getting good results 
After we amplify our DNA we do another round of qPCR. This time, we compare the Input to the IPed sample. Normally we find that the results are similiar to the IP vs IgG qPCR.
That's about it really. It seems to be working fine and we're getting good results
Clare,
Thanks for the response. Just to clarify, i believe that your 'IP sample' is one precipitated with antibody to protein crosslinked to your gene of interest, and IgG sample is precipitated without that specific antibody (IgG only). When you see enrichment, how much do you typically notice? What method do you use? And after amplification what type of Ct values are you seeing for each? Do you also compare these to the IgG sample after amplification?
Sorry to bombard you with questions. There is so much variation in ChIP lingo I want to make sure I understand what a successful practitioner such as yourself is doing.
CB
Clare,
Thanks for the response. Just to clarify, i believe that your 'IP sample' is one precipitated with antibody to protein crosslinked to your gene of interest, and IgG sample is precipitated without that specific antibody (IgG only). When you see enrichment, how much do you typically notice? What method do you use? And after amplification what type of Ct values are you seeing for each? Do you also compare these to the IgG sample after amplification?
Sorry to bombard you with questions. There is so much variation in ChIP lingo I want to make sure I understand what a successful practitioner such as yourself is doing.
CB
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"successful practitioner" ? hehehe 
Yes - the IP is the sample with the antibody - IgG is just IgG. Our enrichment varies between samples (I am using patient samples). At our worst we see 2 cycles, at our best 5-6cycles. But that would depend on what primers you are using too 
From memory, our CT values for our IP are about 25? That's using 6 mill cells/IP and then eluting in 20ul after purification and using a 1:5 dilution of that. And no, we don't compare to IgG after amplification. We did at first though, to make sure we were getting similiar results...everything looked fine so we don't do that anymore
Good luck!
Clare