RT-Q PCR primer design question - (Jan/31/2012 )
So i am going to be doin Q-PCR. I am going to be using oligoDT for conversion of the mRNA to cDNA.
I am using probes and not dye for the q-PCR reaction.I am using Roche primer design assay to design assays (forward and reverse primers and probes for each gene)
Given the fact that oligoDT may not reach the 5' end of mRNA (as it only moves ~1KB), should i be designing primers/probes to be at the left or right end of the output sequence??
i was told by someone in my lab to design them as close to the right hand side as possible (presumably the 3' end), but as i put the cDNA sequence into the assay design program should it not be to the left hand side (5' end)??
Any help would be appreciated as i am getting confused.
I use primer3 software (available on net) to design primers for me. It selects a few set of primers and I test a couple of them. Usually I get one out of 3 to work properly.
Are you using Universal Probe Library Assay Design Center?
If so, find the mRNA reference sequence (NCBI accession) of your gene (that way it can design intron spanning assays) and then look in the table for the best assay near 3' end of mRNA. Introns will be shown there so if you know how long the 3' exons are (you can consult Ensembl) you will know exactly where to look for the assay.
But if you gene doesn't have much exons, there may not be suitable assay. In that case I would design intron spanning SYBR assay using primer3. The Roche application can of course design assays that don't spann the intron, but I don't know if that's a good idea.