Primer design from mRNA or DNA - Confused about what sequence to use for design of GSP and pPCR primers (Nov/19/2008 )

I'm a bit confused about the whole when-to-use-what-template to design primers to make gene specific primers(GSP) for RT-PCR and to make primers/probes for qPCR.

Now, since our target for the first strand synthesis in RT-PCR is the mRNA molecule, should our GSP then be designed to be complementary or the reverse complement to the mRNA sequence? When we do our RT step, we're creating a cDNA strand, but does RTase synthesize the cDNA from 3-5' or 5-3'; will it be reverse complement or just complementary to the mRNA?

i.e. I found an mRNA sequence in GenBank, I want to make a gene specific primer for my RT-PCR step and then I also want to make primers and probe for my qPCR. I don't know if I need to plug the mRNA sequence directly into a primer design program to make primer and probe and then independently make another primer to be my GSP (and when I make a GSP, do I only need a forward or do I need a forward and reverse or what?) OR if I need to take the GenBank sequence and make a reverse complement or simply a complement to give me the cDNA which I would then put into the primer design program.


Basically, I'm confused about how the mRNA sequence accessions in GenBank are formatted (do they read 5-3' or 3-5' or are they actually cDNA, as there are no Uracils in the code?), which way I'm synthesizing my cDNA strand from my mRNA, whether I get two cDNA from this first strand synthesis or just one and when to use what sequences for what primers.

Sense strand, anti-sense strand, this doesn't make any sense to me! Very confused!

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The accessions stand for 5'-3' mRNA sequences ( sense ones those that are translated )
If you put these sequences in any primer program the antisense primer wil synthesize your cDNA. the sense one will form the accession sequence.

İf you are going to perform RT-PCR you better control the product of the primers in a gel. You might get more than one band which is not good for SYBR detection.

The probe can be antisense to the accession sequence or it can be sense to the cDNA.

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Thanks! I wanted to make sure I wasn't going to try and design a primer that would start amplifying in the wrong direction for the first step!



We aren't going to use SYBER green, but in any case, yeah, splice variants or other unwanted bands would be a problem. I'm going to blast the amplicon and sequences to hopefully design something that will not give multiple products, but when I'm trying to design a multiplex with 5 genes (yeah, we'll see if that is even possible!) I don't know if I'll be able to get all the parameters to match on top of that. I'll also do a melt curve on the products to see if I have specific amlifications. Thanks for the help!

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