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
Step 2. Optimize siRNA Delivery to Maximize Gene Knockdown and Minimize Toxicity
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
Step 4. Examine Biological Impact of Silencing Target Gene(s)
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
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.