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Primer design in the promoter region

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Primer design in the promoter region

Postby ChIP » Sun Aug 15, 2010 3:51 pm

I hope, somebody of you can help me out with this:
I want to do a ChIP assay with an antibody against acetylated histone h3. I did a cDNA microarray before and now I want to check if the genes I found there are really regulated by histone acetylation. Thats why I want to do quantitative realtime PCR after the ChIP. The problem is (correct me if I am wrong) that I now have to design primer in the promoter region. And I have no idea how I can do this. Actually I do not even know how to search for the promoter region :oops: . I googled for 2 days now, so I am desperate... :cry: Actually I am not very good in designing "normal" primer...so if you write an answer please do it as if I am in my first semester :)
Thanks
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Postby mqbsspw4 » Sun Aug 15, 2010 6:34 pm

Why do you use the promoter region? If you looking gene expression you need to do it on cDNA using Q-PCR if you want to make a reporter construct then you want to PCR out the promoter region and put it in front of a reporter gene such as GFP or luciferase.
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Re: Primer design in the promoter region

Postby ChIP » Sun Aug 15, 2010 7:25 pm

I already know that the expression of my genes is changed because of the cDNA microarray and subsequent QRT-PCRs and Western blots. Now I want to know if it is because of altered acetylation on the promoter region itself or other mechanisms. Thats why I am using ChIP to extract the regions that have an altered acetylation pattern and after that I need to detect them by QRT-PCR.
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Postby JackBean » Mon Aug 16, 2010 6:17 am

what exactly are you refering to by ChIP? To my knowledge, in ChIP-on-chip you have DNA and immobilize proteins, so that's probably not, what are you doing...
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Re: Primer design in the promoter region

Postby ChIP » Mon Aug 16, 2010 7:02 am

Yes, I immobilize the histones to the DNA, but I am not doing a Chip-on-ChIP, which would be another microarray and certainly the easiest way, but too expensive for my boss. I stop after the Chromatinimmunoprecipitation, extract the DNA and from these pieces I want to do a QRT-PCR.
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Postby zerhos » Tue Aug 17, 2010 2:17 am

if you know the genes sequence,you can isolate the promoter region working on genomic contig
for desig optimized primers i usually use the tool Netprimer.
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Postby JackBean » Thu Aug 19, 2010 6:01 am

I see, you have the antibodies on chip and bind DNA with modified histones, rigth? And wanna know the DNA sequence.
As zerhos wrote, you should be able to get the 5'-UTR from some database, depending on your organism used.
Then there is question, how large your DNA pieces are, because if it was broken between your primer annealing regions, you would not get any signal ;)
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Re: Primer design in the promoter region

Postby ChIP » Thu Aug 19, 2010 7:18 am

I am working with mouse cells. My pieces after shearing are about 300kb. I talked to my boss again and we decided to design primer in the region 300bp from the starting codon. I now know that there is "dcode" where I can look for the 5'UTR, but if anyone of you knows a database that is easier to handle, I would be happy if you tell me.
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Postby JackBean » Thu Aug 19, 2010 7:32 am

as zerhos said, you can always look for the genomic cotigs on NCBI.
Or look into specialized database (I don't know, which one is official of mouse genome, I guess this one http://www.informatics.jax.org/ or you can go to http://www.biomarks.co.uk/ under Vertebrates/Rodents and pick your favourite) and look, whether you can download UTRs
E.g. here http://www.informatics.jax.org/javawi2/ ... &key=53713 go to sequences, choose the genomic and on rigth side you can choose, how long flanks you want ;)
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Postby JackBean » Thu Aug 19, 2010 7:33 am

or, depending on number of your sequences and your Bioinformatic skills, you can look whether they have FTP server and get the sequences from there
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Re: Primer design in the promoter region

Postby ChIP » Thu Aug 19, 2010 8:16 pm

Thank you very much! I will try that. I do not have any bioinformatical skills... But I will spend some time with the databases you recommended. Thank you for that!
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