ChIP data analysis - Calculating enrichment (May/20/2011 )
Hello,
I am trying to calculate ChIP enrichment and have been advised as follows:
1) Quantify DNA in input and IPed samples and then use the same amount of template from each in a qPCR experiment.
2) Calculate enrichment as 2^(Ct IP - Ct input)
The advantage to this method is that it controls for differences in DNA recovery after the ChIP. However, my concern with this method is that pools of IPed DNA can differ depending on the source (e.g. two sample treatments), so it's possible to get values that appear different between two samples even if the amount of IPed target in the pool is the same. Does this make sense?
Does anyone out there have experience in calculating enrichment this way? I did get pretty different DNA readings even between technical reps, so I would like to be able to control for the variable, bu I'm not sure if it's possible. Any dvice would be most welcome!
Thanks!
MJH
i have the same question!
thanks
My method: calculate %Input of IPed material, subtract %Input of IgG from %Input of specific IP, compare your %Input to the IgG corrected %Input from a positive and negative control region.
I wouldn't recommend measuring your DNA amounts and adding "similar template amounts" for my PCR I add 4-9ul of IPed material same for my "input control"
I use 1%Input to normalize my IP values
In reality if your doing your ChIP the same way every time you shouldn't have to correct for DNA recovery abberations
MJH on Fri May 20 18:45:33 2011 said:
Hello,
I am trying to calculate ChIP enrichment and have been advised as follows:
1) Quantify DNA in input and IPed samples and then use the same amount of template from each in a qPCR experiment.
2) Calculate enrichment as 2^(Ct IP - Ct input)
The advantage to this method is that it controls for differences in DNA recovery after the ChIP. However, my concern with this method is that pools of IPed DNA can differ depending on the source (e.g. two sample treatments), so it's possible to get values that appear different between two samples even if the amount of IPed target in the pool is the same. Does this make sense?
Does anyone out there have experience in calculating enrichment this way? I did get pretty different DNA readings even between technical reps, so I would like to be able to control for the variable, bu I'm not sure if it's possible. Any dvice would be most welcome!
Thanks!
MJH
I think your problem here (if I am understanding it correctly) is that you are determining the amount of DNA you have in your input and your IP'ed sample after your do the IP? This sounds like a bad idea as you probably won't have a whole helluva lot of DNA in your IP sample and are likely to get highly variable readings, if any at all. What I did is after mixing together my crosslinked DNA sample into the binding buffer I removed 1% of it as my input. Then you process the input alongside your IP sample (from the elution step onwards) and when you run your qPCR you just load the same volume of input and IP sample. Then you use that to calculate %IP taking into consideration that your IP sample is diluted 1/100.
MM