Ambion's Recommended Procedure for siRNA Hairpin Design
The following recommendations for siRNA hairpin design and cloning strategy are made based on research by Ambion scientists. The first step in designing an appropriate insert is to choose the siRNA target site by following the steps described under "General Design Guidelines" above.
For screening, we typically test four siRNA sequences per target, spacing the siRNA sequences down the length of the gene sequence to reduce the chances of targeting a region of the mRNA that is either highly structured or bound by regulatory proteins. Because constructing and testing four siRNA expression plasmids per target is time-consuming, we find it much easier to screen potential siRNA sequences using PCR-derived siRNA expression cassettes (SECs). SECs are PCR products that include promoter and terminator sequences flanking a hairpin siRNA template and can be prepared with Ambion's Silencer Express Kits. This screening strategy also permits the rapid identification of the best combination of promoter and siRNA sequence in the experimental system. SECs found to effectively elicit gene silencing can be readily cloned into a vector for long term studies. Ambion scientists have determined that sequences that function well as transfected siRNAs also function well as siRNAs that are expressed in vivo. The only exception is that siRNA sequences to be expressed in vivo should not contain a run of 4 or 5 A's or T's, as these can act as termination sites for Polymerase III.