Hi Trond,

The trick is in splicing. GLUT1 (= SLC2A1) has quite a few exons and introns. After transcription introns are deleted during the splicing process. To construct the PCR primers, you need to pick the region, which you want to amplify. To perform RT-PCR, you first need to reverse transcribe your mRNA into cDNA. The cDNA contains only exons, but not introns.

So, the first part (primers, hitting both DNA and cDNA (from mRNA)) -> pick a primer pair, hitting an exon region.

The second part (only DNA) -> pick the intron region, or exon + intron.

The third part (only cDNA (from mRNA)) -> in one of the primers, pick the area of exon overlap, so that it will be complementary only to spliced cDNA.

However, keep in mind, that there are also certain recommendations regarding the length of the primer (usually around 20bp), GC content (50% is the best) and melting temperature (60-70 degrees).

Good luck!

Best,

Michail

Trond Bærheim

Position each of your primers at two exons to identify gDNA and RNA using the same primer pair.

You probably need to identify two exons that are common to all the alternative splice variants of your gene. The attached sketch may be useful.

The above answers are more complicated than necessary. You can design ONE pair of primers that will give different sized products in gDNA and cDNA. Just think about what regions are the same in gDNA and cDNA and what regions are different.