Hi Parisa,

It sounds like you are trying to normalize amount loaded between each sample. When we have performed ChIP (Using Millipore EZ-ChIP), we instead calculate the percent enrichment versus input for each sample, then perform analysis. To do this, save 1% of the supernatant after preclearing and save while you immunoprecipiate the samples. Then reverse crosslink input and IP samples and purify DNA for qPCR.

Remember when you perform qPCR that the amount of input DNA is 1% of total, so subtract 6.644 cycles from Ct, then compare to IP samples.

Using this method, if there is systematic DNA loss during purification, it should occur to the input as well as the IP samples.

Hope that helps!

Good luck!

Dear Jason,

Many thanks for your answer. The method looks interesting. However, I am not so clear about the protocol. So this will be a relative quantification? Why do we always consider 6.644 cycles?

For the q-PCR, I always use 1 µg of DNA. That might not represent 1% of my total DNA.

I will be grateful if you can help me about these.

Many thanks in advance

Kind regards

First you have to determine your yields in order to know you have enough for the number of IP reactions you plan to perform. If you already know the necessary number of cells, and have optimized the shearing conditions, quantification is not necessary unless you use different chromatin samples (different cells) and want to use similar ammounts in the IPs.

In this case you can take a sample of 10-50 µl from the sheared chromatin, decrosslink, isolate the DNA (phenol chloroform method) and check its concentration. (Practically you can measure the DNA concentration of the sample you have purified to load on the gel to check the fragment size).

If you use the same chromatin for different IPs then you don't need to quantitate, because you use the same amount for each IP.

For the relative quantitation of your ChIP results, its best to relate them to the input, just as Jason mentioned. If you take 1% the volume of the chromatin you use for the IP, the calculation of your result will be:

% recovery from input= 2^(Ct sample- Ct input)

here we assume that the PCR has efficiency of 2.

What do you mean you use 1 µg of DNA for the qPCR? Do you mean 1 µl of eluted DNA or?

Dear Teodora, 

Many thanks for your answer. I always quantify my DNA by nanodrop. I use 1µg of DNA for q-PCR as that is the amount that I use for cDNA while performing mRNA expression analysis. I can try it once the way you suggested me. The thing is that I am working with brine shrimp and many parts need to be optimized yet. Even if some of the the kits and protocols from human works well with my organism, but optimizing a correct protocol is not so easy.  

Many thanks for your suggestion. 

Kind regards

Parias