第二,Firstly of all, you should know what you want to dectect.
for example, one gene has 2 spliced forms (transcript variants), but contain differenent exons, the first variant contains exon 1, 3, and 4, the second contains exon 1, 2, and 4. so if you want to detect the total level of this gene, you should design the primer to clone the sequence of exon 1 or 4, if you want to dectect just variant 1, you should design the primer based on exon3. others just like this.