RT-PCR primer design

Question

[Fatemeh Nabizadeh]

I want to design a primer pair for a specific transcript variant of my gene of interest. Is there a way to find out the sequence differences among all transcripts? Should I use alignment tools?

I also used primer blast in NCB‌I for the specific Ref seq accession number, but there are unintended pairs in the results.

How can I do that?

[Hamid Ghaedi]

It seems you know the solution. I would say yes, doing an MSA would be insightful in terms of knowing where at the sequence to pick for primer design to make sure that the PCR amplicon would be generated only from the intended transcript variant.

[Fatemeh Nabizadeh]

Thanks for your response. MSA is calling me :)))