１.Confirm results with a second or third siRNA to the same target. 　　One of the best ways to increase confidence in data from siRNA experiments is to independently use two or more siRNAs to a single target gene. Different siRNAs to the same gene with comparable gene silencing efficacy should induce similar changes in gene expression profiles or phenotypes. Any changes induced by one siRNA and not the other(s) may be attributed to off-target effects.

２.Allow for at least 2 nucleotide mismatches with all off-target genes.

The rules for siRNA specificity are not yet fully defined. Some reports suggest that even a single nucleotide mismatch in the middle of an siRNA can abolish its activity (1,2). In contrast, another report indicates that siRNAs can silence non-target genes containing as few as 14-15 consecutive complementary nucleotides (3). Therefore, until we reach a better understanding of siRNA specificity, it is best to allow for at least 2 nucleotide mismatches between an siRNA and all closely related nontarget genes. The algorithm developed by Cenix Bioscience, and used in Ambion"s validated and pre-designed siRNAs, incorporates a stringent specificity check.

３.Titrate your siRNA.

Nonspecific silencing effects may be seen when an siRNA is transfected into cells at concentrations of 100 nM or higher (3-5). However, this non-specific effect is mitigated when siRNAs are used at lower concentrations (<30 nM). To ensure target specificity, therefore, it is best to titrate the siRNA and use it at its lowest effective concentration.

４.Choose a highly effective siRNA sequence. 　　Using highly effective siRNAs will maximize target mRNA reduction and minimize the possibility of off-target effects by allowing the use of lower siRNA concentrations in the RNAi experiments. The algorithm developed by Cenix Bioscience, and available from Ambion for siRNA design, accurately predicts potent siRNA sequences.

５.Use a scrambled siRNA control.

A scrambled sequence should be used to discount any changes to the gene expression profile that may result from the siRNA delivery method. To serve as a negative control, it is best to ensure that the scrambled siRNA is not complementary to any gene in the target organism.

６.When using siRNA populations, confirm results with individual siRNAs.