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Design strategy for real-time RT-PCR, how to decide on what gene of interest and


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#1 Ddkb

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Posted 26 July 2011 - 08:56 AM

Sorry if this has been asked before, but the more I read it seems the more I get confused!
I'm trying to design a strategy to use real time RT-PCR to look at viable cells in my samples. I'm interested in mRNA (since it degrades fast) as my template. The problem is that the bacteria that I'm targeting is a little unknown. There's a complete genome in NCBI and since rpoB gene is used for a similar assay targeting different bacteria is used, I think I'll go with that. But how do I found out more gene's of interest if this rpoB is not working? Also I'm not sure if it's a single copy gene, how do I figure that out? I was thinking blasting the gen sequence against the whole genome? Does that sound logical?
Next is primer design. Can I use the sequence that is published in NCBI, how do I know what is the right orientation for the mRNA? Because the way I understood it (but maybe I just don't understand it!) mRNA can be both from 5' to 3' but also from 3' to 5' ??? So how do I know which one I'll need for my gene of interest?
I've read that it's better to use intron spanning primers if you're targeting mRNA, so my next question is how do I know what/where the introns are? And also on that note I've read that it's better to design a primer close to the 3' end because that's where mRNA starts degrading. Is that right?
Sorry if the questions are a little scattered, but that's how my mind is currently as well! I would really appreciate any help with this, since I'm completely new in this area.

Thanks

#2 Trof

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Posted 27 July 2011 - 07:12 AM

Can't help you with the first questions, because I don't work with bacteria and have no knowledge about viability testing by RT-PCR.

If you have reference sequence in NCBI you usually use it for primer design, yes. You don't need to know the mRNA orientation but that should be also mentioned on the Gene page of your gene and species of interest on NCBI (the subsection Genomic regions, transcripts, and products). But as I said, you don't need it to do PCR, because you have primers for both strands, the first RT-PCR cycle will create the second strand of cDNA and from there on you have two strands and they amplify exponentially. Just use the published "mRNA" sequence as a template for primer design (in applications like primer3 or PrimerBLAST).

Use of intron spanning primers is widely recommended for distinguishing cDNA (transcribed mRNA) from genomic DNA, but unless you work with Archea bacterias you cannot design them, because bacterial genes doesn't have introns. So you must treat your samples with DNase and check with RT- control.

It's true the mRNA starts degrading from 3' in eukaryotes (5' end is protected by GTP cap) but I'm not sure if is this true for prokaryotes too.

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#3 Ddkb

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Posted 27 July 2011 - 08:19 AM

:) Thanks Trof! That's very helpful!

Can't help you with the first questions, because I don't work with bacteria and have no knowledge about viability testing by RT-PCR.

If you have reference sequence in NCBI you usually use it for primer design, yes. You don't need to know the mRNA orientation but that should be also mentioned on the Gene page of your gene and species of interest on NCBI (the subsection Genomic regions, transcripts, and products). But as I said, you don't need it to do PCR, because you have primers for both strands, the first RT-PCR cycle will create the second strand of cDNA and from there on you have two strands and they amplify exponentially. Just use the published "mRNA" sequence as a template for primer design (in applications like primer3 or PrimerBLAST).

Use of intron spanning primers is widely recommended for distinguishing cDNA (transcribed mRNA) from genomic DNA, but unless you work with Archea bacterias you cannot design them, because bacterial genes doesn't have introns. So you must treat your samples with DNase and check with RT- control.

It's true the mRNA starts degrading from 3' in eukaryotes (5' end is protected by GTP cap) but I'm not sure if is this true for prokaryotes too.






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