RT-PCR Primer design - which region? - (Sep/28/2005 )
Hi, I'm just starting out on designing my first PCR reaction. I've got a suitable set of primers designed by primer3. However, I've been told that primers from the 3 prime non-coding end of the gene are the best ones to use. Could anyone tell me if this is true, and if so, why?
I don't understand why the non-coding end, but if you use poly-T to reverse transcribe, the closer your primers are to the poly-T tail, the better likelihood that your amplicon will be reverse transcribed.
I don't know if I like the "non-coding" part at all, you can overcome the whole problem by using random nonamers for the reverse transcription step, this way the RT initiates at many places within the RNA and even primers at the 5' end of the RNA are likely to have an amplicon available.
Well the best place to design the primer will be a region that you are sure will be in the mRNA sequence. The coding sequence region Would be the best region to design primers to.