I guess there are different ways to approach this problem so I’ll just explain what I’ve been doing. Firstly I do the qPCR on my ChIP and Input samples before amplification (I use the Bio-Rad MyIQ and the standards curve method). I calculate the percentage of the Input DNA that has been ChIP’d using the amount of lysate used, the volume I elute the DNA in, and the amount I dilute the DNA prior to qPCR.

For example I use 60ul lysate for the Input DNA, elute the DNA in 50ul, dilute 1:1000 for qPCR and use 5ul in my qPCR reaction. For the IP I use 300ul lysate, elute in 50ul, dilute 1:5 for qPCR and use 5ul in each qPCR reaction. So I’ve ended up using 1000 times more lysate for each IP qPCR reaction than for each Input qPCR reaction. So percentage input ChIP’d = (IP qPCR value/1000)/Input qPCR value x 100%.

I usually ChIP a few percent of the Input DNA in the experiments I’m doing. I am using a myc antibody to ChIP an epitope tagged protein in a yeast strain I’ve made. As a control I use my myc antibody for ChIP with lysate from a wild type yeast strain (I guess this is equivalent to using non specific IgG). In one experiment I got values 2.34% input ChIP’d for the experiment and 0.02% input ChIP’d for the wild type control.

After amplification I did similar calculations to the one above (knowing how much I diluted the DNA for amplification etc…) to get values for the percentage input ChIP’d.

In an attempt to get enough material to hybridise, we initially ended up over amplifying the DNA (in the above experiment the percentage input ChIP’d went down to 1.22% after two rounds of amplification and the wild type control increased to 0.28%).

We then decided to try different cycle numbers (20, 25 and 30 cycles) in our amplification step to see where the background started to increase to an “unacceptable” level. For us 25 cycles was OK but by 30 cycles we had a significant increase in the value for the percentage of control ChIP’d. So to get enough material to hybridise we fixed the amplification at 25 pcr cycles and used more ChIP DNA for the pcr.

Hope this helps. I'd be interested to hear how other people approach this problem. 

I'm doing some ChIP assay as well but I'm very confused about the QPCR set up and also the enrichment calculation methods. I hope I can get some help from you guys.

1) To carry out QPCR after ChIP, do we need to put in the same amount of unChIPed DNA (input DNA) and ChIPed DNA for comparison?

2) For the standard curve, a paper by Haring et al. suggested that it can be performed with the cross-linked and sonicated DNA (I think it's input DNA then). What DNA do you guys use?

3) Enrichment calculation method?? Haring et al. has a good summary of the ChIP assay and they suggested "% input" or "relative to control sequence" are better options. What do you recommend?

Thank you for your help in advance!

Cheers,
Wen