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[Sugan]

I am designing primers for RT-PCR. I should be designing primers spanning the exon junctions to avoid DNA contamination. The Primer-Blast software has options for only on primer to span an exon junction. Is it necessary to have both primers to span the exon junctions or shall I go with Primer-Blast results? What can be done for sequences without exon information? Please Advise.

[Trof]

One primer spanning an exon junction is enough.

As for the other question, do you have a reference sequence without exon information but in other databases like Ensembl you can find where the exons are, or you have a sequence and you just don't know where the exons start and end?

In the first case, you can still design exon spanning primer if you limit the primer position within the junction (not sure if this is possible in primerBlast, but it can be done in primer3) but you can do this only with one junction at a time, just pick one or few and design primers for each separately, IMHO pretty laborious.

If you don't know anything about the exons, then you probably can't do anything with it.