I’m designing primers for SYBRGreen-based qPCR (using the software Primer3).
I’ve tried to check the specificity of the primers by blasting them. However, one of the potential off-targets given by Primer-BLAST is:
It should still work, but that will decrease your PCR efficiency, and if you are working with multiple strains/subjects, the effect will vary and not be competely correlate with your control. So I would avoid that particular combination if at all possible.