I'm currently trying an X-ChIP-qPCR experiment to determine the H3 occupancy at some specific loci in yeast cells. I was wondering what internal control I should use. I found a lot of articles of people normalizing the IP/IN signal of the primer of interest with th IP/IN signal of a control position where the occupancy of H3 is constant, but only in plants. I had a hard time finding the same in yeast, although it seems to be a good control to me. Is there a point I'm missing?
Best normalization is percent enrichment in IP fraction versus input. I don't think it is standard practice to normalize to a locus where histone occupancy is thought to be constant. If you want to include this as a positive control to make sure your ChIP is working well, that is reasonable, but I'm not sure it is a good method for normalization. In theory, this type of normalization could correct for experiment-to-experiment variations in ChIP efficiency, but this would similarly be reported by simply checking enrichment over input.
Well, thank you for your advice. I quite always use the %IP/IN but I wanted to introduce a control for the variations, from one sample to the other, of the efficiency of the IP itself.
So, I think I'll find some regions with well-known H3 occupancy and just see if I got what I expect - as you suggested.
Hi Aria,
couldn't you use cell number for normalization and Input as quality control for fixation/DNA isolation etc?
Since every cell contains a full genome, you could simply use a defined cellnumber for each sample! Since Yeast usually grows in suspension cell number can be optained pretty easily.
Best Kai
Hello!
I rely on my IN for that, since my protocol is highly reproducible so far. And as you said, it's a good control of the lysis and chromatin preparation efficiency.
However, working with the H3 antibody is all new for us, and I would have liked a control, not of the amount of total DNA, but more of the antibody efficiency and it's reproducibility from one sample to the other. I actually want to know if the variations I observe are specific to the gene I study.
For example, in inducible conditions I have a very low H3 occupancy at the A gene. But is it due to the induction of the expression of the A gene, or is the H3 signal low all over the genome - for some unknown reasons ?
What internal control should I use? I need a gene whose expression does not respond to stress or nutrients. In the literature, I found a lot of this kind of control in experiments about plants, but not in yeast.
What did I miss? What do you use as control? I imagine I should go for primers in a promoter with a strongly positioned -1 nucleosome for example, but which one? I guess there are some typical control I could try to use in my conditions - instead of testing randomly.
I hope it's clearer, and I apologize for my english. Thank you very much for your interest in my question.
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