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I have a gene sequence with 15 alternative splicing forms. I want to design a siRNA for 1 isoform, I select the exon intron junction that is unique for this isoform, and I must design siRNA for this sequence or this unique isoform, but this sequence has a similarity with another sequence when I blast it, also this sequence doesn't have features for siRNA design. With this condition how can I design siRNA for my sequence?

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  • $\begingroup$ Can you show how the sequences align? A rough illustration is sufficient if you do not want to share the details. Plus why are you targeting intron-exon junction? You can simply target the exon that is retained in the desired isoform. $\endgroup$ – WYSIWYG Aug 3 '16 at 18:47
  • $\begingroup$ i aligne these sequences with clustal omega, and also i have this schematic picture in the literature.my desired isoform is the smallest sequense between another isoforms with two exons,and all of the isoforms have both of above exons.therefore i have to select exon intron junction , that its the uniqe sequence. $\endgroup$ – zh.a Aug 4 '16 at 12:55
  • $\begingroup$ Have you extended the siRNA to 25 bases (usually considered the longest) and moved it up and down the junction to try to find a more unique sequence? i.e. have at least 7 bases on each side of junction, but vary it from 7 to 18 on one side and see if you get more unique blasts. I assume you are blasting RNA and not DNA? $\endgroup$ – akaDrHouse Aug 4 '16 at 17:56
  • $\begingroup$ Can you show the alignment? siRNAs have to be perfectly complementary and the 5' pairing is the most crucial. There are other rules too. Also, please tell what were your BLAST parameters. Edit your question to include all necessary details otherwise it would not be possible for us to help you. $\endgroup$ – WYSIWYG Aug 4 '16 at 18:45