Hi Experts,
I am trying to do ChIP qPCR assay. So I followed the protocol from EMD Millipore/Sigma Aldrich kit for ChIP assay.
According to the protocol, at the end of ChIP, just use 2 uL of eluted DNA for PCR/qPCR. However, I was thinking of normalizing the DNA amount that I will use for PCR/qPCR. I tried using Nanodrop to measure DNA concentration, but the values are way too low (almost nothing for positive control, negative control (from the kit) and target antibody-bound DNA). Should I just follow the kit (by not measuring DNA concentration) and directly use 2 uL (or equivolume) of DNA for downstream?
If this is not the best method, may I ask what is, and for what reason?
Thanks for your advice!
Hey edward,
After performing ChIP and pulling down the DNA sequences through antibody I suggest instead of using nanodrop , you must quantify and normalize your output DNA through absolute qPCR with standard curve which gives you precise and exact concentration of your sample.
Because SYBR much more sensitive to small concentration of DNA or even a single molecule and standard curve of known template gives you better normaliztion of your sample.
I faced the same problem while performing chip. the problem is with DNA elution procedure i guess. I use standard phenol chloroform extraction method to elute DNA after reverse cross-linking. for me, adding 3M sodium acetate along with overnight isopropanol precipitation of DNA could improve yield significantly.
Dear Edward
Have you tried using Qubit or Xpose for quantification and qualification?
Regards,
As answered above I would go for qPCR or very sensitive detection methods, such as Qubit or Picogreen. Nanodrop won't be of any use here and even quite sensitive devices such as Agilent BioAnalyzer with a DNA high sensitivity chip are not able to give you a concentration of your ChIP-ed DNA. qPCR, though, requires that you know at least one potential target site of your DNA binding protein to design PCR primers for your positive control (a desert gene or some constitutive heterochromatin domain can be useful to design primers for a negative control). Good luck!
Thanks for your answers. I will definitely try Qubit or Picogreen. How come DNA analyzers are not able to read and measure concentration? It's boggling to me that the kit I am using assumes that every sample will have relatively similar concentration.
The fact that most analyzers are not able to give a reliable concentration of ChIP-ed DNA relies mainly in the proportion of target sites compared to the whole genome, and the fact that your DNA is sheared into 200-500 bp fragments. Except if you start from a large number of cells, you'll precipitate only minute amounts of DNA.
Now, as for the kit starting with the same volume for every sample, it isn't the same thing as assuming you have the same DNA concentration in all your samples, provided the good controls are performed. Usually when performing ChIP-qPCR, one uses some control IgG to give an idea of non specific DNA precipitation, so that's a first level of "normalization". Then one also usually compare precipitated DNA with a reliably concentration-measured input, which also gives a ratio or an idea of ChIP efficiency, which could be different among samples. Lastly, you may (should) compare between genomic loci where you know (or hypothesize) that your factor is binding and loci where you're sure that it's not the case, so that you can calculate some kind of enrichment value for your ChIP. Then even if your starting concentrations are different, they will be taken into account by the different comparisons (mainly sample vs input and then positive vs negative loci).
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