# 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

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

Hi,

I'm not much experienced but your data seems to be fair. When you compare negative control region to your region, you have a significant enrichment. I think you do not have to expect a value bigger than your input because you immunoprecipitate a tiny population of your input(at a given time period not all fragments that contain your region are occupied, and you do not crosslink all of them, and you precipitate only some portion of your TF crosslinked to your region).

Thanks