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Primer design and alternative transcripts


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#1 assembler01

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Posted 26 March 2014 - 12:36 PM

My lab had some old real time PCR primers for some of the genes we're looking at now. I've been double checking to make sure they were optimally designed by BLAST and checking the exon/intron structure. For some genes there are a few or even a huge number of potential alternative transcripts (for example, I've been looking at Prkaa2, Slc2a4 among others). I don't see any published evidence for most of them so I assume they were just predicted by whatever algorithm NCBI/Ensembl or whoever uses. Western blots for them typically show just one band so some of them can't be protein coding or perhaps even exist at all. Then I find the primer binding sites for most of the published/validated primers and I notice they almost always bind some exon that is not in every transcript. What do you typically do in those situations? Do you just ignore any of the putative transcripts if there is no experimental evidence? Do you design primers that would bind most or all of the putative transcripts?



#2 bob1

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Posted 30 March 2014 - 12:55 AM

I don't have any references for it, but it does seem that sometimes so called "pseudogenes" are expressed - however, if the peptide sequence produced is the same as the original target does this matter functionally?



#3 bob1

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Posted 30 March 2014 - 12:56 AM

I don't have any references for it, but it does seem that sometimes so called "pseudogenes" are expressed - however, if the peptide sequence produced is the same as the original target does this matter functionally?






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