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Old 08-04-2014, 05:51 PM   #1
cfourps
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Location: West Lafayette, IN

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Default Alternative splicing versus incomplete splicing?

In cDNA generated from poly-A reverse transcription of RNA, is it possible that PCR will detect incompletely spliced mRNA? How does one differentiate between something that is a legit isoform and an incompletely spliced molecule?

On amplifying using primers binding to the 5'UTR and the 3'UTR, I am seeing a faint longer band in addition to a band at the size I expected based on the predicted mature mRNA.
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Old 08-05-2014, 01:13 AM   #2
dpryan
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Yes, that can happen and the best way to determine if it's a different isoform or just incompletely spliced would be to look and see if the reading frame changes. If so, you likely have intron inclusion. If not, well, you still might but there's no real way to rule that out other than perhaps seeing if the result encodes a protein and if the protein works (though one could successfully argue that either not producing a protein or producing a non-functional protein could both be results of a properly spliced transcript).
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