Generate?siRNA?Populations?with?Dice

The use of small interfering RNAs (siRNAs) to induce targeted gene silencing in mammalian cells offers researchers a powerful tool for analyzing gene function. Ideally one would like to work with individual pre-designed or validated siRNA sequences (see The Easiest Route to Guaranteed Silencing ). However, when pre-designed or validated siRNAs aren't available, considerable time and money can be saved by generating a population or cocktail of siRNAs by enzymatic digestion of a long dsRNA. Populations of siRNAs elicit gene silencing effects comparable to individual validated siRNAs, and usually work on the first try (once delivery is optimized), without the need to design and screen individual siRNAs.

Mimic the Endogenous RNAi Pathway

Ambion now offers a new tool for RNAi research: The Silencer™ siRNA Cocktail Kit (Dicer) . Dicer (or one of its homologues) is the enzyme involved in processing long dsRNAs into siRNAs in the endogenous RNAi pathway in eukaryotes. Dicer produces 21-23 bp cleavage products that are considered to be an optimal length for RNAi induction. For these reasons there is considerable interest among researchers in the use of Dicer to generate siRNA cocktails for RNAi induction. The Silencer siRNA Cocktail Kit (Dicer) and the stand-alone Dicer enzyme provide ideal tools for this application.

Effective Gene Silencing with Dicer siRNA Cocktails

The Silencer siRNA Cocktail Kit (Dicer) was used to reduce the expression of target genes. This reduction was compared to knock down elicited by chemically synthesized siRNAs. Data is presented in Figure 1 demonstrating that siRNA cocktails generated by Dicer were as effective as chemically synthesized siRNAs. Under these experimental conditions, the siRNA populations generated using the Silencer siRNA Cocktail Kit (Dicer) reduced GAPDH and Ku-70 protein expression by 98% and 85%, respectively.

Figure 1. RNAi Induction by Dicer-generated siRNA Cocktails and Validated siRNAs. siRNA mixtures prepared with the Silencer ™ siRNA Cocktail Kit (Dicer) and individual Silencer ™ Validated siRNAs, both targeting GAPDH and Ku 70, were transfected into HeLa cells at a final concentration of 50 nM each. 48 hr after transfection, cells were analyzed for Ku 70 or GAPDH protein expression by immunofluorescence using anti-Ku (p70) and anti-GAPDH antibodies (B) . To generate the bar graph (A) , fluorescence of a single microscopic field was quantitated using MetaMorph software (Nikon). The negative control siRNA cocktail was prepared from the ß-gal gene.

Specific Reduction of Target Gene Expression

To address concerns about potential nonspecific effects associated with the use of siRNA populations for RNAi experiments, the effects of Dicer-generated siRNA cocktails targeting the human STAT3 transcript on the expression of several STAT gene family members was examined. As seen in Figure 2, STAT3-specific siRNA cocktails suppressed the expression of the STAT3 gene only, without affecting other members of the STAT gene family. This data shows that siRNA populations generated using recombinant Dicer are effective and specific mediators of gene silencing.

Figure 2. Specificity of siRNA Cocktails When Targeting a Conserved Region of a Gene Family. HeLa cells were transfected with siRNA cocktails derived from the human STAT3 gene generated using the Silencer ™ siRNA Cocktail Kit (Dicer). 48 hr after transfection, the cells were harvested and analyzed by real-time RT-PCR for target mRNA levels. 18S rRNA levels were used to normalize STAT gene expression. Percentage of remaining STAT gene expression is shown here.

A Complete Kit for Generating siRNA Cocktails

Most commercially available kits for producing siRNA cocktails with Dicer include reagents for preparation of 5 siRNA cocktails. The Silencer siRNA Cocktail Kit (Dicer) contains reagents to generate 7 dsRNA cocktails so that users can prepare 5 experimental siRNA cocktails, as well as both positive and negative control siRNA preps, which are crucial to RNAi experiments. The kit includes reagents for transcription and cleanup of long dsRNA, as well as Dicer enzyme and reaction buffer. Also included are templates for transcription of a negative control siRNA and a positive control siRNA targeting GAPDH.