RNAi: A Four-Step Workflow
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.
- High sensitivity western blot kit—detect femtogram levels of proteins with x-ray film or CCD-based imaging
- Image immediately or up to 24 hours later
- Compatible with multiple membrane types, including PVDF, nitrocellulose, and nylon
The new Western-SuperStar Immunodetection System can be used for many applications involving immunodetection of proteins immobilized on membrane. With its improved signal intensity and rapid detection, the Western-SuperStar System is well suited for applications such as detection of RNAi-mediated protein knockdown (Figure 2, bottom of page). This kit includes everything you need for western blot detection, such as blocking agent, secondary goat anti-mouse or anti-rabbit antibody-alkaline phosphatase conjugate, wash and assay buffers, ready-to-use chemiluminescent substrate, and enhancer for nitrocelluose membranes—even the recommended blot incubation and detection containers are provided.
The Western-SuperStar System uses a next generation chemiluminescent substrate to deliver an extremely sensitive western blot detection method. After a 5 minute incubation with the SuperStar™ Substrate, blots can be imaged immediately. In addition, the signal persists with low background and consistent sensitivity for imaging at any timepoint up to 24 hours after blot development.