RNAi: A Four-Step Workflow

RNA interference (RNAi) is the biological mechanism by which small interfering RNA (siRNA) induces gene silencing through targeting complementary mRNA for degradation. This cellular process is revolutionizing the way researchers study gene function. For the first time, scientists can quickly and easily reduce the expression of a particular gene in nearly all metazoan systems, often by 90% or greater, to analyze the effect the gene has on cellular function. The ease of the technique, as well as the wide availability of high-quality kits and reagents for performing RNAi experiments, has driven its incredibly rapid adoption by the research community. This article outlines the workflow for a typical RNAi experiment using siRNAs.

Step 1. Obtain Effective siRNAs

To obtain gene silencing, potent and specific siRNAs are crucial. In addition, good experimental design dictates that at least two effective siRNAs be used in the experiment to confirm that the observed effects result from knocking down the gene of interest.

Step 2. Optimize siRNA Delivery to Maximize Gene Knockdown and Minimize Toxicity

Efficient, reproducible siRNA delivery is essential for successful RNAi experiments. The best siRNA delivery protocol provides good gene knockdown (i.e., effective delivery), while maintaining an acceptable level of cell viability (i.e., low cytotoxicity).

Negative control siRNAs are needed to identify potential non-specific effects on gene expression caused by introducing any siRNA. Easy-to-assay positive controls are also needed for optimization of transfection conditions, control of siRNA delivery, and as downstream assay controls.

Step 3. Test siRNA Silencing Efficiency

Because siRNAs exert their effects at the mRNA level, the simplest and most sensitive assay for siRNA validation relies on real-time RT-PCR to measure target transcript levels in cells transfected with gene-specific siRNAs versus negative control siRNAs.

Step 4. Examine Biological Impact of Silencing Target Gene(s)

Assays that measure the effects of gene silencing include morphological, enzymatic, biochemical, and immunological assays. siRNAs affect target mRNA levels, but phenotypic changes are usually due to reduction of protein levels. siRNA-induced silencing at the protein level is typically measured by western blotting to correlate the observed phenotype with the level of knockdown induced (see sidebar, Western-SuperStar™ Immunodetection System).

To confirm that an observed phenotype is due to RNAi, it is also useful to perform time course and siRNA titration experiments.

Using siRNAs to Delineate Gene Function

To help you get started in RNAi, Applied Biosystems has created an online workflow which describes step-by-step how easy it is to use our products to accelerate gene function analysis.

Scientific Contributors
Albana Mihali, Kathleen Skaare, and Corinne Miller • Applied Biosystems, Bedford, MA


Figure 2 (Sidebar). Silencer® siRNA-mediated Knockdown of Survivin Detected with the Western-SuperStar™ Immunodetection System. HeLa cells (2.5 x 105 cells/well; 6-well plate) were transfected with either Silencer siRNA (Survivin, 30 nM; siRNA ID #2554) or Silencer Negative Control #1 (NC, 30 nM) using siPORT™ NeoFX™ Transfection Agent (5 µL/well). Cell lysates (24, 48, or 72 hrs after transfection) were electrophoresed (10 µg protein/lane; 4–15% Tris-HCl gel) and transferred to a PVDF membrane. The Western-SuperStar System was used to detect survivin, then after stripping the blot, GAPDH. The blot was exposed to X-ray film (5 sec) or a CCD imaging system (1 sec) immediately after incubation with the SuperStar Substrate. NT=non-treated HeLa cells.
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