- siRNA duplexes should be composed of 21-nucleotide sense and antisense strands, paired to have a 2-nt 3’ overhang (a TT overhang tends to work well).
- A particularly effective target motif is NAR(N17)YNN, where N is any nucleotide, R is an A or a G, and Y is a C or a U.
- Target region should be selected from a cDNA sequence beginning 50 to 100 nucleotides downstream of the start codon.
- Target cDNA sequence should have around 50 percent GC content.
- Avoid more than three consecutive identical nucleotides.
- Secondary structure of the target mRNA does not appear to affect silencing.
- Selected siRNA sequences should be Blasted against EST libraries to ensure that only one gene is targeted.
Source: The siRNA User Guide: http://www.mpibpc.gwdg.de/abteilungen/100/105/sirna.html