gene-specific Primer for qRT-PCR - (Sep/13/2007 )
I'm new on qRT-PCR. I would like to design primers which spann exon-exon boundaries. I'll work on a qRT-PCR one-step kit with Sybr. My problem is, that i don't find any primer-design programs, which design the primers so that BOTH (forward and reverse) span the border....
Maybe you have some ideas for me? I hope you can help me!
Many thanks in advance!
Well, you can either purchase pre-made primers from a company or design them yourself.
I do the latter. Basically I use Invitrogen's VectorNTI (now free for academic/federal labs) combined with the UCSD Genome Browser. Grab sequence from GenBank for a mature mRNA for the gene of interest, use UCSD's BLAT server to BLAT it against the human genome, identify the exon-intron junctions and design three primers for both a pre-mRNA specific and a mature mRNA specific transcript. (for example, 1 left primer in an exon, and then either a primer in the next intron for pre-mRNA or a primer in the next exon for mature). Now of course, this assumes that your intron is sizable. If you are doing real-time pcr with a 45s extension cycle (common) then pick a pair of exons that flank an intron of at least 1 - 2 kb in size. Basically you wont get amplification of your pre-mRNA signal becuase the pcr cycle time is not long enough. Of course, make sure you validate by running your PCR reactions out on a gel in addition to using your ABI or BioRAD qPCR device.
This was pretty short; if you would like a more detailed explaination.. i'd be happy write it up.
thanks for your reply, I will now try this program!