How to choose siRNA when BLAST results show high similarity with other gene - (Jan/03/2006 )

Hi Dear all,
I am still a little confused about designing the siRNA although I learned a lot from this Forum.

I use the Online siRNA Design Tools to design my siRNA, I can get several siRNAs. But when I click the BLAST link, they always show they have 100% similarity with many other genes. I know that will cause off-target effects.

Could you please give me some suggestion about the blasting result?

Thanks a lot!

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Hello,

If those hits are on mRNA sequences of other genes, that should be a concern. Definitely 100% homology with other genes is unacceptable. Even partial homology can be tolerated, the homology should be restricted only to the 3' end of the antisense strand of the siRNA, which means don't allow the seed sequence of the siRNA to have targets on other genes.

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Thank you very much!

When I do the blast, the following seq. always show up:

1. gi|46309674|gb|AC100208.16| Mus musculus chromosome 7, clone RP23-59J14, complete sequence
Length=280763

Score = 30.2 bits (15), Expect = 13
Identities = 18/19 (94%), Gaps = 0/19 (0%)
Strand=Plus/Plus

Query 1 AAGTCCGCAGAAGGGTTAA 19
||||| |||||||||||||
Sbjct 220030 AAGTCAGCAGAAGGGTTAA 220048

2.
>gi|78190197|gb|AC154426.2| Mus musculus BAC clone RP24-199A21 from chromosome 14, complete
sequence
Length=210568

Score = 28.2 bits (14), Expect = 51
Identities = 14/14 (100%), Gaps = 0/14 (0%)
Strand=Plus/Plus

What is that mean? It seems those are not genes, should I consider them especially those with 100% homology when I design siRNA?

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Based on the blast results you gave it is hard to tell whether the hits are on a gene or not because those sequences are large fragments of genomic sequences. You should do a blast against only the RefSeq database.

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How is RefSeq database different from the rest?

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Check NCBI website, you will find out what RefSeq represents. In short, Refseq represents the most correct version of gene coding sequences.

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