I have a problem with the logic of an RNA IP normalization. I am comparing two RNA binding proteins tagged with GFP, one is an isoform of the other where I mutated one binding site. My IP is done with anti GFP antibody. At the end I recover RNA. So, my question is how to normalize?
My thinking is: taking input is not relevant because this does not take into account IP efficiency. What I do is I quantify the total RNA after IP, I then run an RT-qPCR on the same amount of RNA and I normalize over an unspecific IP mRNA. I don't know if it is a good way but I think this takes into account the input which is highly variable because I work on live animals and not on cell culture. And I hope that this takes into account IP efficiency?
I have the same problem and I am thinking about using a spiking control. I would use a RNA-fragment which can be further used for cDNA sysnthesis and qRT and I would put it in all samples (same amount) right befor you do the IP. The problem I see ,that you have to be sure that the control mRNA you use should be unspecifically bound in the same manner by both proteins.
yes the idea is good but the probleme is still as you say if both protein will bind with same manner.
One issues i try is to normalize the amount of RNA after qPCR on the amount of protein IP without taking internal non specific gene.
hope it will be accepted by reviewer.....
I am having very similar issues. I found this RIP kit guide from Sigma that talks about qPCR calculations (on page 15)... I think I may try to use this concept, but instead of having a non-specific antibody maybe adjusting against a housekeeping or endogenous mRNA that should not be IP'd. Let me know what you think of this. Not sure if this is allowed either?
http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Bulletin/1/ripbul.pdf
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