I have made knockdown of a gene by stable transformation. I have few transformants to screen for best silenced line. The total length of the mRNA is 1440 bps. The region of the sequence I have used to clone as a hairpin construct is from 175 bp to 1035. My question is to screen the transformants by real time RT-PCR, where should I design my primers, should it be in 5´(1-175bp) or 3´(1040 to 1140 bp) prime region of the sequence. If the primers are with in the region of 175 bp to 1035 bp does effect it my result. Which is the best region to design primers for real time to screen for the best silenced line.
Edited by Nirvana09, 03 December 2010 - 01:50 PM.