The genome has already been sequenced and I am looking to design primers. I've tried using primer BLAST on NCBI website but find it confusing.
You'll have to add in some additional parameters for qPCR:
1. primers MUST be in mRNA sequences - no promotor, no introns, no intergenic
Even better, design primers that span an exon-exon junction so they can't amplify a gDNA product
2. you want short products - 100-150bp is ideal
3. design several pairs to test. Many won't have high enough efficiency for qPCR.
4. You need to include a "housekeeping" steady-state expression control pair of primers. Check the literature and order a few from a recent publication. Test them yourself anyway.
Good luck!
Use BLAST mRNA Coding DNA sequence (CDS), Then you can put it in online software (Primer3 software https://bioinfo.ut.ee/primer3-0.4.0/) or design manually.
In software, you have to put annealing temperature and follow 22 to 26bp primer sequence sizes to avoid nonspecific amplification.
PCR product-180 to 200bp or less than 200bp is good
Use housekeeping gene for normalization.
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