siRNA Design Guidelines 【Ambion】

3. Design appropriate controls.

A complete siRNA experiment should include a number of controls to ensure the validity of the data. The editors of Nature Cell Biology have recommended several controls (2). Two of these controls are:

• A negative control siRNA with the same nucleotide composition as your siRNA but which lacks significant sequence homology to the genome. To design a negative control siRNA, scramble the nucleotide sequence of the gene-specific siRNA and conduct a search to make sure it lacks homology to any other gene.
  
• Additional siRNA sequences targeting the same mRNA. Perhaps the best way to ensure confidence in RNAi data is to perform experiments, using a single siRNA at a time, with two or more different siRNAs targeting the same gene. Prior to these experiments, each siRNA should be tested to ensure that it reduces target gene expression by comparable levels.
  

Ambion's siRNA Target Finder

Use our online target finder to find potential sequences based on the design guidelines described above. Simply paste your mRNA sequence into the window and this program will scan your sequence for AA dinucleotides. A report is generated indicating the position of the AA dinucleotide, the 21 base target and the corresponding sense and antisense siRNA oligonucleotides. siRNA targets can then be sent directly to one of our kit-specific design tools or subjected to a BLAST search by clicking on the appropriate link below the target of interest.

Alternatively, the Whitehead Institute of Biomedical Research at MIT has a publicly available siRNA design tool that incorporates additional selection parameters and integrates BLAST searches of the human and mouse genome databases. See http://jura.wi.mit.edu/pubint/http://iona.wi.mit.edu/siRNAext/ (registration required).

Specific Guidelines for Designing siRNA Hairpins Encoded by siRNA Expression Vectors and siRNA Expression Cassettes

Researchers who initially reported the use of siRNA expression vectors to induce RNAi had different design criteria for their inserts encoding the expressed siRNA. Most of the designs had two inverted repeats separated by a short spacer sequence and ended with a string of T's that served as a transcription termination site. These designs produce an RNA transcript that is predicted to fold into a short hairpin siRNA as shown in Figure 1. The selection of siRNA target sequence, the length of the inverted repeats that encode the stem of a putative hairpin, the order of the inverted repeats, the length and composition of the spacer sequence that encodes the loop of the hairpin, and the presence or absence of 5'-overhangs, vary among different reports (3-11).

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.