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Designing primers for qPCR

When designing primers for qPCR, which sequence is better to consider, CDS or full gene?

gene
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updated by Biostar Biostar • written by casley_queiroz • casley_queiroz

Do you with full gene mean also intronic sequences, or CDS + UTR?

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written by WouterDeCoster WouterDeCoster

qPCR for cDNA I assume? Do you expect introns in your mature transcript/cDNA? Or are you talking about qPCR on genomic DNA?

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written by WouterDeCoster WouterDeCoster

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.

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written by casley_queiroz • casley_queiroz

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?

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written by WouterDeCoster WouterDeCoster

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).

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written by casley_queiroz • casley_queiroz

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