BLAST of shRNA sequence - (Dec/06/2011 )

Dear all,

I want to blast my shrna sequence to see if it is specific. I used megablast (NCBI) and nucleotide collection as database. I got many many things other than my gene of interest.

Could you please tell me, how I can determine if my shrna is specific enough to my gene of interest? Is "total score" a good representative for specificity? Should I use nucleotide collection as database or refseq-rna (this one gives less hits)?

Thanks

-Ambinlab-

To make your BLAST results easier to read, just use the sequence in the shRNA for the antisense strand of the siRNA as query (exclude loop, and flanking sequence in the shRNA) and RefSeq as the database. Your target gene should be at the top of the hit list. there may be partial matches to unrelated genes, that is fine if the matches are not significant (>80% homology) especially in the seed region.

-pcrman-

Thanks pcrman,

what is seed region? sorry I know it is a silly question....I got what I attached to this post. it is around 100 other genes that are matching my antisense sequence. does it mean this shrna has lots of off targets?

-Ambinlab-