Hi Terrie,
the easiest explanation for these results is that you're not using isoform-specific oligonucleotides for you qRT-PCR. I would double check if you For and Rev are annealing properly to all your isoforms and they are producing an amplicon with the same length.
In case either the For or Rev primers are not annealing on all the isoforms you will have to correct that by re-designing them. Also if the amplicon size is different you might not have the same amplification efficiencies and get not conclusive results or no amplification at all in case is too big (it could happen in isoform 2 for instance).
In case your oligos are fine however, there might be some issues with your constructs, which could be more difficult to solve.
Hope it helps
Francesco
Have you checked that the plasmids have the correct inserts? Also, are the inserts cDNA derived or do they include exons and introns?
I agree, you should check ID of your inserts before transfection and if this was done check whether the analysis tool adequately designed and working (primer design, read out, especially!!! controls positive and negative would help!!!).
A more academic and less easy explanation could be over activation of splicing apparature in your human kidney cell system (embryonic), but this is far away from probable!
- Badges
- Science method