ChIP-Qpcr Relative to Input Issues - (Jan/01/2009 )

Hello,

I am trying to get my head around expressing ChIP-QPCR results as a percentage of input. I know there have been several discussions on this but I am still a bit confused. Below is an example of an recent experiment. For the sake of clarity, I previously ran a 10 fold dilution series in my input samples. The efficiencies looked decent. So...

Question:
My main question is if my Transcription factor Ab, in my gene of interest is showing a ct value higher than my 1% input, but much lower than my IgG control. Is this real binding, and can we actually call this "enrichment" even though the actual amt of DNA that pulled down with the transcription factor Ab is less than the 1% of input?

As a negative control, I did compare these (input, Tf Ab, IgG) to another region where the transcription factor is though Not to bind. There was a clear difference when graphed out, but all of the transcripton factors compared to input were less than 1. (sample=0.29% to cntrlregion=0.03%)

I hope my questions were clear. Just trying to have confidence if I had a successful ChIP. If the primer efficiencies are acceptable, input seems very arbitrary.

I have been used to ChIpping with Histones and they have always given me >1% relative to input, but now using transcription factors for the 1st time, I am starting to second guess because they are lower than 1,


To get data I took used POWER(2,ctsample-ctinput) to get relative to input values.

Here are some values
Sample region where I am interested in transcription factor binding

CT value
Input (1%) =25.5
TF =27.3
RbIgG =33.1

POWER(2,ctsample-ctinput)
Input (1%) = 1
TF = 0.295
RbIgG = 0.005



Negative cntrl region where there is no or little suspected transcription factor binding

CT value
Input (1%) = 26.3
TF = 31.4
RbIgG = 35

POWER(2,ctsample-ctinput)
Input (1%) = 1
TF = 0.03
RbIgG = 0.002
Any help would be appreciated. If repeated, is this reasonable to say we have tf binding to the sample region?

Thank you