RT-PCR to amplify ORFs - (Aug/20/2008 )

Hi,
I have a sequence and have found the ORFs and have designed primers to run RT-PCRs. How will running these RT-PCRs show me which ORF actually transcribes the protein? As long as my primers cut where they are suppose to, I will get a band on my gel, no?

What is the point of running the RT-PCRs to amplify the ORFs?

thanks
Loussan

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Your RT-PCR will only work if your ORF is actually transcribed. If it is not, then no RNA is made, hence you can't make cDNA from it and you will not get a product by RT-PCR. Be careful though, because you may get a product off of the genomic DNA. You should have designed your primers spanning an intron so that you can tell the difference (your RT-PCR product will be smaller), and you should make certain to DNase treat your RNA prep to get rid of genomic DNA contamination.

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Thanks for the help.

so if the ORF is a coding region for a gene then it should be transcribed and I will see product. If my ORF is incorrect and is not a coding region I will have no product?

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Yes. With the caveat that if your promoter is tissue specific, developmental specific, or only induced under certain circumstances, then you may not see a product even though the ORF is valid. If you've identified your ORF, you should blast it against various databases for ESTs or cDNAs before you even do RT-PCR. That will tell you if there is data already present which suggests that your ORF is correct. An absence of such data will not say for certain that the ORF is incorrect, but if it is present, then it will help you to determine if you are on the right track.

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