Designing ChIP primers
Posted by Joana Carvalho, on 4 September 2014
Hello everyone,
Recently I got assigned with the task of designing good primers for ChIP. My supervisor advised me to use the Primer BLAST tool from NCBI together with AmplifiX to get some computer-generated primers and at the same time test some I designed myself. Problem is we were discussing yesterday and eventually we came up with a question: should the primers include the binding sites for the transcription factor we are interested in or there is no problem if they don’t as long as the binding site is sufficiently close to the region amplified by the primers?
In the meantime I already looked for some info online and the idea I got is that it’s not mandatory that the designed primers include the binding site region. However there is not much more info on the subject (at least that I could find…)
So I would like to ask to people that are more familiriazed with this technique: in your opinion what is the best?
Thank you so much in advance!
Cheers! ;)
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Hey Joana,
I am going to assume you are asking for help designing primers for a model system that is not mouse or human and that you are using Sybr and not Taqman (if any of this is wrong, let me know).
Since primers are pretty cheap, I have usually designed a couple sets – how you are describing should work fine. I would also suggest designing a control primer set that is a couple kB away from your binding site (maybe against the 3’UTR of a close gene, or an intron far from the binding site), in addition to an unrelated control genomic region. That way, you can control for your sonication and your antibody specificity. Lots of websites offer primer design – I have used SciTools on the IDT website. I put in my genomic region of interest and choose the best sets of primers the computer designs. I would also check my primer sets with PrimerBlast. You don’t necessarily need to design primers that overlap your binding site, but I wouldn’t go more than 200 or 300 bp away from it (depending on what your sonication looks like). I work in Xenopus and sometimes the genome is really A/T rich, making primer design right at my region of interest difficult. I’ve successfully used primers that were designed for regions adjacent to my binding sites of interest a number of times.
Another control I usually do is to check how the primers amplify in QPCR using genomic DNA. You want to make sure they make one specific product, with one peak in the melt curve, before even trying them with your ChIP samples.
Let me know if I can answer any other questions and good luck with your experiment!
Hey Christine!
Actually I am designing primers for human, and yes, I am using Sybr Green.
I have designed a couple of pairs already using different genomic regions with potential binding sites (both in the promoter and in the first intron of the gene) and also others in neighbouring regions that do not contain any binding sites to use as internal controls. I also did a set of primers in a different unrelated gene that has already been proved to interact with the transcription factor I am interested in and that I will be using as a positive control.
Now all that is left to do is order and test them!
Thank you so much for these tips, they were really useful!
Good luck for your experiments as well!
Joana