ChIP quantification (qPCR) - (Feb/23/2006 )

It's already quite some time that I'm puzzled about how to quantify the enrichment in ChIP.

Some people use: 2^(Ct,mock - Ct,IP)
whereas others use something like: %total = 2^(Ct,input - Ct,IP) x %input amount (as %total)

What is actually the best method? Is there a method that takes both Ct,input and Ct,mock into account? I use the first method right now.

Suppose you have two conditions:

Ct,input1 = 25 Ct,IP1 = 29 Ct,mock1 = 33
Ct,input2 = 26 Ct,IP2 = 30 Ct,mock2 = 33

Would you conclude 16-fold enrichment in condition 1 and 8-fold enrichment in condition 2?? What about the input values? Do they not count?

Suppose you have:

Ct,input1 = 25 Ct,IP1 = 29 Ct,mock1 = 33
Ct,input2 = 25 Ct,IP2 = 29 Ct,mock2 = 34

Does this mean 16-fold and 32-fold enrichment? Why should the Ct,mock values differ anyway?

As a matter of fact, I have had this last situation a few times, but I don't think I can just conclude that there is a difference in enrichment between the two conditions.

Take a look here: Genome Biology 2005, 6:R64 (doi:10.1186/gb-2005-6-8-r64)

and here: Received November 18, 2002; Revised March 24, 2003; Accepted April 23, 2003

but there´s a programm (Target Analysis Quantification (TAQ) software) involved in this way to quantitate.
If you don´t have some fancy software, try this:

for your antibodies:

[ct (targetpromoter, enriched fraction) - ct(targetpromoter, INPUT)] /[ ct (negativecontroll, i.e. a region within the genome, which is not bound by your Ab, enriched) -ct(negative, INPUT)]

and accordingly the same with your -Ab controll.

then you can make a histogramm with this formula and set a treshhold of say 3 cycles.

So for your antibodies you should get some values like 3 or above and for you nonantibodycontroll
you should get something below 2.

cheers,

thenotsowIseone ( I know I know, was simply too tired as I tipped in this stupid name )

ups my fault the second paper is:
Nucleic Acids Research, 2003, Vol. 31, No. 12 3257±3266