# ChIP qPCR data analysis? - (Feb/13/2008 )

I have been running some ChIP assays to demonstrate binding of some transcription factors to a promoter, but I am having trouble analyzing the data. I was planning to use the delta-delta Ct method, but since my negative IgG control doesn't amplify at all, I'm not sure how to do the calculation. I get nice Ct values for my input and some of my transcription factors and my negative IgG control is always undetectable, which seems good on the surface. I am running 55 cycles and get get Ct values around 23-25 for my input and around 30-33 for some of my transcription factors. Do I just use 55 for the negative control? This seems arbitrary as I could run 60 cycles and then use 60. Should I switch analysis methods and use %input instead? I'm not sure what the accepted presentation is for this type of data.

Thanks,

Kurt

Which detection system are You using (sybr-green, taqman, ...)?

I suggest You to use the %input method to present the data, at any rate. It's a well known way to present enrichment of protein binding to/loading on DNA.

Anyway, comparisons among data gained from different antibodies would bias Your results: different antibodies may have (actually, do have) different efficiency.

GL!

Thanks,

Kurt

I agree that you should express your data as % of input. However, what is most important is that you compare your enrichment at your region of interest to enrichment at another region where you don't expect your TFs to bind. Enrichment itself (however you calculate it) doesn't have much inherent meaning unless you can show a baseline (enrichment at a region of the genome where your factor doesn't bind) and show that your enrichment is above this baseline at your region of interest. Alternatively, you could knockout, or knockdown, your factor of interest to show the baseline.

Hello, could anyone further explain how to express the results as % of Input. Thank you in advance.

What method are you using for detecting PCR products (e.g. real time, EtBr stained gel, etc).

Hello, I would like to perform a Real Time PCR to measure the enrichment of acetyl H3 in cells treated with HDACi. I've seen people who represent the data as CtInput-Ctsample but other people use different methods. I'm not an expert so, at this point, I don´t know which method I must use.

Thanks

Thanks

If your primers are close to 100% efficient then 2^(CtSample-CtInput) will work. If not, you can always run a standard curve using dilutions of your input DNA, find the equation for the curve, and quantitate your sample using this equation.

Thanks

If your primers are close to 100% efficient then 2^(CtSample-CtInput) will work. If not, you can always run a standard curve using dilutions of your input DNA, find the equation for the curve, and quantitate your sample using this equation.

Hi！KPDE, I also have such a question.

As you suggest, I run a standard curve using dilutions of my Input DNA( Can I use a genomic DNA to do this standard curve?), and I got the equation for the curve (such as y=-3.07x+22.107 ,y stands for CT, x stands for the dilution fold ) but I still can not get the quantitation of product by negative-control region-primers (these two primers works well in Input DNA,but no amplification in IP fraction) , its ct is N/A .So what can I do to do the calculate? Thanks!

Another question,how to evulate the enrichment ratio? Above what number can we have confidence that our region is enriched by this TF protein?

Have a great day!

buyi

Thanks

If your primers are close to 100% efficient then 2^(CtSample-CtInput) will work. If not, you can always run a standard curve using dilutions of your input DNA, find the equation for the curve, and quantitate your sample using this equation.

Hi！KPDE, I also have such a question.

As you suggest, I run a standard curve using dilutions of my Input DNA( Can I use a genomic DNA to do this standard curve?), and I got the equation for the curve (such as y=-3.07x+22.107 ,y stands for CT, x stands for the dilution fold ) but I still can not get the quantitation of product by negative-control region-primers (these two primers works well in Input DNA,but no amplification in IP fraction) , its ct is N/A .So what can I do to do the calculate? Thanks!

Another question,how to evulate the enrichment ratio? Above what number can we have confidence that our region is enriched by this TF protein?

Have a great day!

buyi

You'll need to be able to see amplification for your IP before you can quantitate. Once you do get good Cts for your IP with primers for your region of interest (and preferably lower Cts than your mock IP) then, to determine if your enrichment is real, you should compare the signal to another set of primers for a region where you don't expect to see your factor bind. Your enrichment is only significant if it is higher at your region of interest than another region of the genome where your factor shouldn't bind.

Thanks

If your primers are close to 100% efficient then 2^(CtSample-CtInput) will work. If not, you can always run a standard curve using dilutions of your input DNA, find the equation for the curve, and quantitate your sample using this equation.

Hi！KPDE, I also have such a question.

As you suggest, I run a standard curve using dilutions of my Input DNA( Can I use a genomic DNA to do this standard curve?), and I got the equation for the curve (such as y=-3.07x+22.107 ,y stands for CT, x stands for the dilution fold ) but I still can not get the quantitation of product by negative-control region-primers (these two primers works well in Input DNA,but no amplification in IP fraction) , its ct is N/A .So what can I do to do the calculate? Thanks!

Another question,how to evulate the enrichment ratio? Above what number can we have confidence that our region is enriched by this TF protein?

Have a great day!

buyi

You'll need to be able to see amplification for your IP before you can quantitate. Once you do get good Cts for your IP with primers for your region of interest (and preferably lower Cts than your mock IP) then, to determine if your enrichment is real, you should compare the signal to another set of primers for a region where you don't expect to see your factor bind. Your enrichment is only significant if it is higher at your region of interest than another region of the genome where your factor shouldn't bind.

I exactly did what you said. But the problem is I get signal for IP sample with primers for my region of interest,but no signal for IP sample with primers for a negative region (no TF binding region) . It seems the signal for IP sample is real,but I don't know how to calculate it. Thanks!

"Once you do get good Cts for your IP with primers for your region of interest (and preferably lower Cts than your mock IP) then---"

Another problem is I do get good cts for my IP sample,but it is no significant difference with mock IP. I used the normal IgG from santa curz. But I think it is not very important,since my IP sample give nearly no signal for two primers （both for regions supposed to be non binding region. And I also get not very good CT from primers for other region I tested ) So I think it is not necessary to try another normal IgG as the mock IP.

thanks!