Imagine a gene produces 2 transcripts, isoform a with exons A-B-C and isoform b with exons B-C-D. I have an RNAi clone that matches uniquely exon A,. The generation of siRNAs from isoform a could potentially target isoform b. But under what circumstances will I observe a knock-down of both isoforms? e.g. Do the isoforms need to be expressed in the same cell? Are there position or size requirements for the shared exons?
I have been unable to find a publication dealing with this issue.
I don’t know of a definitive answer in the literature, let alone someone testing this, but according at least to my vague recollections we should be able to make some inferences from what we’ve been told of the role of RNA-dependent-RNA-polymerase (RDRP) activity. It seems to me that in the situation you describe, with the unique exon being 5’ to the shared exons, dsRNA specific to the unique exon should not be expected to target transcripts lacking that unique exon, either in the same cell or in other cells: at least as I recall, the generation of additional siRNAs beyond the homology provided by your siRNA should be dependent on RDRP activity and should go from 3’ to 5’. Though I admit I haven’t looked to double-check what was initially published, let alone what’s been published more recently.
If on the other hand you target shared sequence or there exists shared sequence 5’ of the unique exon, at least in theory you’ll be out of luck: you should expect at least the possibility that RNAi’ing the unique exon will produce dsRNA and siRNAs 5’ of that exon, and target both isoforms. Cell-specific expression seems unlikely to help prevent this, because of sid-1.