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Table 1 Guidelines for siRNA design

From: Towards in vivoapplication of RNA interference – new toys, old problems

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
  1. Rules for the design of synthetic siRNAs according to Tuschl and coworkers [27] and some further considerations for vector-based small hairpin RNA (shRNA) expression are given. A collection of links to small interfering RNA (siRNA) design tools on the internet is provided. nt, nucleotide; UTR, untranslated region.