I have a question regarding ChIP-qPCR normalization. I am very familiar with qPCR and gene expression analyses but very new to chromatin immunoprecipitation qPCR. When performing gene expression, the variation in RNA extraction from sample to sample is corrected by total RNA quantification to ensure an equal amount is loaded in each RT reaction, followed by normalization by the use of housekeeping genes at the qPCR level.
For ChIP, I didn't find any details regarding correction of the input amount of DNA in the qPCR reaction. From the numerous protocol that I red, it seems like people don't care and perform their IP on a fixed volume of ChIP DNA (no matter what the total DNA quantity as judged by spectrophotometer) and/or use a fixed volume of purified DNA at the qPCR level. As the preferred method of analysis is to simply process the raw Ct value and expressed it as a % input followed by comparison across multiple experimental conditions, I am wondering how one can account for the variability from sample to sample in such a long protocol without virtually any normalization?
Can anyone with strong hands-on experience with ChIP-qPCR comment on this please?
Hey JP
I am not sure if I understood your question but here it goes.
Usually I control for the amount of DNA I am IP in two instances.
After I sonicate my samples I take a small volume out (5 to 10ul) and do a quick reversal of xlinking and DNA purification. At this point I quantify the total amount of DNA I have in my sonicated samples (this is also valuable to determine how many ug of DNA you need per ug of antibody for the IP portion of the protocol).
Once I have the DNA quantified I use the same amount for all the samples (I will take the sample with the least amount of DNA and dilute out all the others to match the same amount).
From here I then control for the volume of Input to calculate the percent of input.
Hi, I've been doing ChIP for a while now and was wondering the same question. From the literature and protocols from various sources I found that you could either measure total protein concentration of your chip lysates and use the same amount each time for an IP. The other way is to reverse crosslinks and quantify the amount of DNA in starting material and use approx 25 ug DNA/IP. I've used the %input method with and without protein quantification and they both gave me the same trend (if thats what you are interested in). Otherwise its really hard to get comparable Ct values from individual experiments.
I also had the same result with - ptn - dna quantification along with %input method. I think the important step is to be sure about the amount of Dna (assuming an IP with the same quantification of starting material) in your Pcr.
Hey JP
I am not sure if I understood your question but here it goes.
Usually I control for the amount of DNA I am IP in two instances.
After I sonicate my samples I take a small volume out (5 to 10ul) and do a quick reversal of xlinking and DNA purification. At this point I quantify the total amount of DNA I have in my sonicated samples (this is also valuable to determine how many ug of DNA you need per ug of antibody for the IP portion of the protocol).
Once I have the DNA quantified I use the same amount for all the samples (I will take the sample with the least amount of DNA and dilute out all the others to match the same amount).
From here I then control for the volume of Input to calculate the percent of input.
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