The RNAi Web: RNAi/siRNA Design



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siRNA Design

Rules for selecting siRNA targets on mRNA sequences:

Targets should be located 50-100 nt downstream of the start codon (ATG).
Search for sequence motif AA(N₁₉)TT or NA(N₂₁), or NAR(N₁₇)YNN, where N is any nucleotide, R is purine (A, G) and Y is pyrimidine (C, U).
Target sequences should have a G+C content between 35-60%.
Avoid stretches of 4 or more nucleotide repeats.
Avoid 5'URT and 3'UTR, although siRNAs targeting UTRs have been shown to successfully induce gene silencing.
Avoid sequences that share a certain degree of homology with other related or unrelated genes.

How to obtain a mRNA or cDNA sequence for target selection:

Before picking siRNA target on the gene of your interest, first you have to obtain its mRNA sequence from a nucleic acids database or sequence accession number as some siRNA design tools can take accession number as input. It is recommended to use the gene's RefSeq from NCBI, since the RefSeq represents non-redundant, curated and validated, thus most correct, sequences. RefSeq mRNA sequences have unique accession numbers which start with NM or XM, followed by 6 digits. For example, NM_123456 (curated mRNA sequence) or XM_0123456 (model mRNA sequence predicted by genome sequence analysis). There are several ways of searching and retrieving a RefSeq.

Search the NCBI Gene database (Entrez Gene) by gene name or symbol at http://www.ncbi.nlm.nih.gov/gene/ and select the right gene of desired organism, go to the page of the gene, scroll down to the "mRNA and Protein(s) " section and look for mRNA sequences with a accession number started with NM or XM.
Search Nucleotide database using Entrez query tool at http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Nucleotide and use Entrez Limits settings to restrict your query to the RefSeq database only
- select "RefSeq" from the "Only from" menu, this restricts the query to the RefSeq collection
- select "mRNA" from the "Molecule" menu, this restricts the query to mRNA RefSeq records

Homology search
The siRNA targets on the mRNA sequence of a gene should not share significant homology with other genes or sequences in the genome, therefore, homology search is essential for preventing off-target effects. Although most siRNA design tools provide BLAST search option, some simply use NCBI's BLAST tools which sometimes are quite slow. Here are some BLAST tools for homology search.

NCBI Blast tool: Nucleotide-nucleotide BLAST (blastn) or Search for short, nearly exact matches
Blat tool on UCSC Genome Website http://genome.ucsc.edu/cgi-bin/hgBlat
Ensembl Blast http://www.ensembl.org/Multi/blastview

Examples of RNAi target selection

References

Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature. 2001 May 24;411(6836):494-8.
Elbashir SM, Lendeckel W, Tuschl T. RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev. 2001 Jan 15;15(2):188-200.
Reynolds A, Leake D, Boese Q, Scaringe S, Marshall WS, Khvorova A. Rational siRNA design for RNA interference. Nat Biotechnol. 2004 Mar;22(3):326-30.
Tuschl Lab, The siRNA user guide.

siRNA Design

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