I'm trying to design specific qRT-PCR probes for the 2 splice variants of my gene-of-interest. This is what they look like:
Variant 1: Exon1-Exon2-Exon3
Variant 2: Exon1-(Exon2, slightly longer)
Basically, the coding region of Exon2 from Variant 2 contains a tiny piece of intron from Variant 1. I designed a probe for Variant 1 across the border of Exon2 and Exon3, so that should be fine. However, my question is, even if I design a probe against the tiny bit of Exon2 that's unique for Variant 2, wouldn't it still pick up signal from the pre-mRNA of Variant 1, which will still have that piece of intron before splicing?
Please advise! I appreciate any comment on this. Thanks much!!