how to convert RNA-based siRNA to DNA-based siRNA? - (Aug/31/2005 )

Hi, everyone. I want to knock-down one gene permanently, so i choose siRNA expression vectors with antibiotic selectable Markers, such as ambion's pSilencer 3.1-H1 puro.

From other's paper, the RNA-based siRNA of my target gene was used and succeeded in downregulation the expression of the gene. I want to use the same target sequence as that paper said.

But the target sequence is 21bp, and the first two nt is not AA, which seems not suitible for siRNA expression vectors(the web recommend choose 19nt after AA).

How can i do? Just choose the last 19nt?

For example: the sense-strand sequences used to target each gene were as follows:
Dcp2 "5'-GTGGCATGTAATGGACATTGC-3' "
Ref; RNA (2005), 11:717-727.
A role for eIF4E and eIF4E-transporter in targeting mRNPs to mammalian processing bodies.

If I synthesis siRNA the sense strand should be 5'-GGCATGTAATGGACATTGCTT-3'.
the antisense strand should be 5'-GCAATGTCCATTACATGCCAC-3',

If i want to construct it into pSilencer 3.1-H1 puro vector, the 19nt I choose should be "5'-GGCATGTAATGGACATTGC-3'.

Is what I think right? Hope for your help.
Thanks a lot.

tracy

Yes, you are right.

One of the early Tom Tuschl rules is to pick siRNA target after a AA dinucleotide. Later it is found that this rule is not a must. Also the overhangs don't necessarily need to be TT.

You can use Ambion's oligo template design tool to design your cloning oligos http://www.ambion.com/techlib/misc/psilencer_converter.html which can save you lot of pain.