qPCR primer design for multiple transcript variants

Question

[Jordan]

Hello, I am a little new to NCBI tools and can't quite wrap my head around primer design on the website. I am trying to design primers to run single-step qPCR for one gene's expression. There are 3 transcript variants, all of which are used for the single-step qPCR. Specifically, I need to design primers common to all 3 transcripts around exon 4. Previous qPCRs have been run as 2-steps (cDNA synthesis then subsequent qPCR) but results have not been good. Can I reuse any of these previous primers for the one-step? If not, how do I set the primer blast parameters to identify primers for all 3 transcripts at exon 4?

Answer 1

[Kevin Blighe]

Presumably, the previous cDNA synthesis step was done for a reason, which may relate to how your target gene exhibits sequence similarity elsewhere in the genome. A few pre-selection / amplification protocols exist for such difficult regions of genome.

You can quickly check the primers' predicted target(s) via the UCSC's in silico PCR tool: https://genome.ucsc.edu/cgi-bin/hgPcr

Otherwise, there are two workflows listed here (for primer and/or probe design:

• PCR Primer or Primer+Probe design
• Designing a Primer

Kevin