Hello Everybody,
I want to design a gene-specific primer for RT-PCR. What do I need to keep in mind?
Thanks a lot
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#2
pcrman
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Posted 29 May 2009 - 02:03 PM
Do you mean doing RT using gene specific primer? I think all RT-PCRs are gene specific.
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Posted 01 June 2009 - 11:14 PM
pcrman, on May 30 2009, 12:03 AM, said:
Do you mean doing RT using gene specific primer? I think all RT-PCRs are gene specific.
I do not mean real time PCR. I want a primer for "reverse transcription PCR. Normally you use oligo dTs or random primers. But the mRNA I am looking for is very seldom. I thought that I can increase the efficiency for that gene by using a gene specific primer.
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leelee
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Posted 02 June 2009 - 03:39 AM
I don't think that pcrman thought you meant real time........... and the RT-PCR reactions I have performed have all been gene specific, it is not an uncommon thing (I would actually suspect that the vast majority of RT-PCR reactions are using gene specific primers rather than the method you were describing....but I could be wrong, it has been known to happen 
)
I have a question for you though, how were you hoping to find your mRNA if you weren't going to use gene specific primers?? How would you know it was the mRNA you were looking for?? I am confused......
If you give us a bit more info about what exactly you will be doing, and any concerns you have, we might be able to help you better.
)I have a question for you though, how were you hoping to find your mRNA if you weren't going to use gene specific primers?? How would you know it was the mRNA you were looking for?? I am confused......
If you give us a bit more info about what exactly you will be doing, and any concerns you have, we might be able to help you better.
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Posted 02 June 2009 - 06:45 AM
If you give us a bit more info about what exactly you will be doing, and any concerns you have, we might be able to help you better.
[/quote]
First, thanks for your answer!
And know my problem.
I am trying to fish the gene of a receptor (not the whole gene, only the complementary sequence to the mRNA, for over expression in a mouse cell line). Therefore I took tissue, isolated total RNA and used the RNA as a template for cDNA synthesis (made by RT-PCR with oligo dT primers because I need a full length clone). Afterwards I used the cDNA for gene amplification by normal PCR (with gene specific primer).
But the receptor, I am looking for, is rarely transcribed and my trials failed.
I thought I can enhance the efficiency of the RT-PCR by using gene specific primers. But in our institute nobody did it before.
[/quote]
First, thanks for your answer!
And know my problem.
I am trying to fish the gene of a receptor (not the whole gene, only the complementary sequence to the mRNA, for over expression in a mouse cell line). Therefore I took tissue, isolated total RNA and used the RNA as a template for cDNA synthesis (made by RT-PCR with oligo dT primers because I need a full length clone). Afterwards I used the cDNA for gene amplification by normal PCR (with gene specific primer).
But the receptor, I am looking for, is rarely transcribed and my trials failed.
I thought I can enhance the efficiency of the RT-PCR by using gene specific primers. But in our institute nobody did it before.
