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mRNA PCR


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#1 scilyn

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Posted 16 July 2009 - 11:01 AM

Hello,

I am trying to make primers for PCR amplification, basically to test if a gene and the transcript is present in our cell types. I am searching for some nucleotide sequences on NCBI, but I am only finding mRNA sequences, not DNA sequences. mRNA transcript would probably be better to amplify to make sure the transcript is there...

Basically, I need to know if I should amplify all the exons, or some of the exons. Or, if I want to figure out quantitatively how much transcript is present would I need to do RT-PCR and then create primers against the cDNA. Is this the same as mRNA? I'm so confused, please help!

Thank you!

#2 cellcounter

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Posted 19 July 2009 - 02:46 PM

Hello,

I am trying to make primers for PCR amplification, basically to test if a gene and the transcript is present in our cell types. I am searching for some nucleotide sequences on NCBI, but I am only finding mRNA sequences, not DNA sequences. mRNA transcript would probably be better to amplify to make sure the transcript is there...

Basically, I need to know if I should amplify all the exons, or some of the exons. Or, if I want to figure out quantitatively how much transcript is present would I need to do RT-PCR and then create primers against the cDNA. Is this the same as mRNA? I'm so confused, please help!

Thank you!


mRNA sequences are deposited in DNA format after DNA sequencing. So, you can use that to design primers against your cDNA (first strand synthesis) for RT PCR. For your purpose, mRNA is cDNA.

You must use mRNA/cDNA sequence for transcript analysis, DNA sequence (~genomic DNA sequence) is not good for this purpose, unless you map it out for where the exons are, and then use only those regions for PCR primer generation.




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