Table 1 Guidelines for siRNA design

General guidelines for siRNA design

Select 23-nt long sequences from the mRNA conforming to the consensus 5'-AA [N19]UU-3' or 5'-NA [N19]NN-3' (where N is any nucleotide)

Avoid targeting of regions that are likely to bind regulatory proteins, such as 5'-UTR, 3'-UTR and regions close to the start site

Choose sequences with GC content between 30% and 70%

Avoid highly G-rich sequences

Design sense and antisense N19 sequences, add two 2-deoxythymidine residues to the 3' ends

Perform BLAST search to exclude potential homology to other genes

Additional considerations for vector-based siRNA expression

Avoid more than three consecutive As or Ts in the targeting sequence

U6 promotor requires a guanine at position +1

H1 promotor prefers adenosine at position +1

Design oligonucleotides containing N19 targeting sequence, a loop forming spacer sequence (often 5'-TTCAAGAGA-3'), followed by the reverse complementary targeting sequence and five to six consecutive thymidine residues for termination of transcription

Add respective restriction sites for cloning

siRNA design tools on the internet

http://www.ambion.com

http://www.qiagen.com/siRNA

http://jura.wi.mit.edu/bio/

http://www.dharmacon.com

http://sinc.sunysb.edu/Stu/shilin/rnai.html