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
- 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
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.
Examples of RNAi target selection
- 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
- Tuschl Lab,