several questions for siRNA target BLAST - (Nov/25/2004 )
I have some questions about how to blast a siRNA target sequence. I write
here what I think the procedure should be. Please tell me what are your
suggestions about it, especially when you think something is wrong with it. Thank you very much.
1. The database should be "EST". If the organism is mouse, the database
should be "EST_mouse". (Is it right?)
2. It is "7" for "word size". But what should "Expect" be? Which option(s)
should be selected for "choose filter"?
3. In the column of "Options", "organisms" is "Mus musculus" if the
organism is mouse. (is it necessary because the database has been
4. After get the BLAST results, click the blue "U" in the right, check if
all the results come from a same gene (your target gene). But for some
results, there is no blue "U" in the right, then how to check if it is the
target gene or not? Moreover, if there are another genes listed in the
BLAST results, how to decide if the siRNA target sequence should be
filtered out or not? Lie on "E Value" or the amount of nucleotides
homologous to the another genes? What is the threshold of "E Value" or the
amount of homologous nucleotides to decide if the siRNA target sequence should be filtered out or not?
I have just begun to work in RNAi and my English is not very good. I hope, and I look forward to get your suggestions as soon as possible. Thank you very much.
I assume that you are talking about using NCBI blast tool, right?
If you have selected a target sequence which is around 19-25 nt, you can use the "Search for short, nearly exact matches". The default word size is 7. If you choose to use the regular blastn program, you can also set the word size to 7. For database selection, you can use refseq_rna because refseq represents well all transcribed sequences. Although you can also use EST database which, however, is very redundant and contains sequence errors.
If you choose to use refseq_rna, specify the organism to mouse. For the parameter of "Expect" just use the default.
If a EST hit doesn't have "U", it means that the EST has not been assigned to any Unigene and probably is a singleton, you can just ignore it. If you get hits on ESTs that belong to another unigene, you have to decide how much homology is toleratable for your RNAi experiment. I think the identity should not exceed 70%.
I would suggest that you start your RNAi adventure by using some siRNA design programs which have the blast feature built-in. Those program will take care of target selection, homology exclusion, and finally returns to you all acceptable siRNA targets on the gene of your interest. Many companies provide such tool such as Dhamacon, invitrogen and ambion. You can find all those program here http://www.rnaiweb.com/RNAi/RNAi_Web_Resou...ools/index.html
Yes, I am using NCBI blast tool. Thank you so much for your help, pcrman. Have a good thanksgiving day!
I have an another question: when I blast, should I use the 21nt (AA19nt) or only 19nt without AA?
Thank you very much.
You can just use the 19 nt sequence without AA.
You are welcome.