When designing primers for qPCR, which sequence is better to consider, CDS or full gene?
Do you with full gene mean also intronic sequences, or CDS + UTR?
Just intron + exon, wihout UTR.
qPCR for cDNA I assume? Do you expect introns in your mature transcript/cDNA? Or are you talking about qPCR on genomic DNA?
Thank you for your support. I got an answer. Ideally designing between an exon + intron + exon. Thus eliminates the risk of losing a amplification if primers were designed in intro, because this would not be the cDNA.
Definitely not in an intron, best having intron spanning amplicon (one primer in exon n and the other in exon n+1) or a primer spanning the exon-exon junction. Are you sure you understand what you are doing?
I think I really understood, I drew one of the primers in the junction of two exon and the other within outher exon (exon 2 or 3 for exemplo).
Okay, I just came across this posts so this might be of interest for you: http://eu.idtdna.com/pages/decoded/decoded-articles/pcr-qpcr/decoded/2016/06/28/use-splice-junctions-to-your-advantage-in-qpcr