I did chromatin extraction using both commercial kit and homemade recipe. By using RNase A and protease K digestion together with phenol-chloroform-isoamyl or on-column cleaning, I then isolated the sheared DNA directly after chromatin extraction prior to the proceeding to immunoprecipitation so as to determine the shear efficiency and quantify DNA using High sensitivity DNA chips from Agilent. From the results, my DNA concentrations were hundreds of pg/uL, but I do not know how to use that to calculate the amount of chromatin.
And the second question is, since I work with chicken samples, should my positive control for ChIP (anti-RNA polymerase II) be specific to chickens, or since RNAP II is highly conserved in eukaryotes, general ChIP-grade Ab should be fine? And for the controls for IP (I used RNAP II and non-immune IgG), should they also be used as the positive and negative controls in the qPCR later, or the qPCR should use genomic DNA as a positive control and use input DNA reverse cross-linked directly from chromatin extraction as reference? And in such ChIP-PCR study, should the reference genes also be used for final calculation?
For the analysis, t is indicated in many protocols that one should use either the percent of input DNA, which is reverse cross-linked from chromatin extraction, or fold enrichment in according to the negative control (e.g. non-immune IgG). However, since the non-immune IgG should not have reactivity to any specific loci, but only provides the information how much background is presented in the product, shouldn't the Ct value be extremely high or even undetermined eventually? If it is undetermined, how could I calculate the fold enrichment then?
Thank you for your time!
Dear Yang Xiao, as for your first question, to put it simply, chromatin is a complex of DNA and the four core histones, and possibly some other tightly associated chromosomal proteins. But the two major components are DNA and the core histones in a roughly 1:1 mass ratio, depending on the length of the linker DNA which can vary between species. So, when you determined the DNA amount, you already know the amount of chromatin. As for the anti-RNAP II antibody, I would believe that you need to check the antigen used to generate the antibody and whether that particular antibody would react with chicken RNAP II. I don't think the RNAPII is that highly conserved in terms of primary sequence. So, you need to use an anti-body specific to chicken RNAPII. For the controls, I think you can use both genomic DNA (input DNA) and non-immune IgG pulldown samples as controls. I think this is also relevant to your analysis, as long as you know what the comparisons are. Hope this would help. Good luck with your experiments.
Hi Hua Xiao ,
Thank you very much for you kind answers! Greatly appreciate it! I actually just tried the protocol including the qPCR. What surprised me the most was that the Ct values of my samples and the input, PC and NC were all fair (samples around 28, NC around 32) with low SD among parallels, while the DNA concentration of those DNA samples were super low from both High sensitive DNA quantification chip (Agilent) and Qubit, with some even cannot be determined. Though it is possible that the concentration would drop below the threshold (10 pg/uL), with such low concentration, I would not expect such Ct values then. It is not possible that there was contamination to all my samples when running qPCR and all with the similar amount of DNA contaminants. Could you please provide any insights what could be the cause of such results?
Thank you very much!
Yang
- Badges
- Science method