Transfection optimization is critical to performing meaningful siRNA experiments in cultured cells
The GAPDH (glyceraldehyde 3-phosphate dehydrogenase) gene is an abundant and ubiquitously expressed “housekeeping” gene that can be manipulated and measured providing two-fold utility for siRNA optimization experiments in cultured cells. First GAPDH gene expression can be readily knocked down in many cell types by delivery of the validated Silencer® GAPDH siRNA. The efficiency of siRNA delivery can be easily monitored by measuring the reduction in GAPDH protein levels in cells transfected with GAPDH siRNA relative to cells transfected with negative control siRNA. Second, GAPDH expression can serve as a marker for identifying cellular toxicity resulting from transfection.
Easy Transfection Optimization
Rapid Measurment of Silencing and Cell Viability
Figure 1 indicates that while GAPDH knockdown was observed for all transfection conditions tested, the best knockdown was observed using 0.6 µl siPORT NeoFX per well. These protein knockdown results mirrored those for mRNA knockdown obtained using real-time qRT-PCR (best knockdown observed for 0.6 µl siPORT NeoFX conditions, data not shown). Since cytotoxicity was a problem at the very highest amount of transfection agent (1.2 µl/well), the best optimal transfection conditions were determined to be 4,000 cells and 0.6 µl siPORT NeoFX per well.
Figure 1. Fast Transfection Optimization Using the Silencer® CellReady™ Transfection Optimization Kit and the KDalert™ GAPDH Assay Kit. 4,000 and 8,000 HepG2 cells were transfected with either Silencer® GAPDH or Negative Control #1 siRNA using a Silencer CellReady siRNA Transfection Optimization plate and varying amounts of siPORT™ NeoFX™ Transfection Agent. Silencing of GAPDH expression for each transfection condition was measured using the KDalert assay. Residual GAPDH activity was determined from the ratio of GAPDH activity in samples transfected with GAPDH siRNA divided by the GAPDH activity in corresponding samples transfected with Negative Control #1 siRNA. Transfection associated cytotoxicity for each of the transfection conditions was also measured using the KDalert GAPDH Assay Kit by comparing GAPDH activity of negative control siRNA transfected samples to that of untreated samples. Identified optimal transfection conditions are highlighted in pink.
Optimized Transfection Conditions Can be Used with Confidence
Figure 2. KDalert™ Optimized Transfection Conditions Silence Five Different Genes. HepG2 cells were transfected with Silencer® Pre-designed siRNAs targeting five different genes in 96 well microplates using the optimized conditions determined in Figure 1 (4,000 cells/well, 0.6 µl siPORT™ NeoFX™/well). Two days after transfection, the mRNA levels in each of the transfected cultures were compared to cultures transfected with Negative Control #1 siRNA using real-time qRT-PCR.
Ambion's Complete Solution for siRNA Transfection
Luis Foncerrada, Kevin Kelnar • Ambion, Inc.
The 3 Steps:
1. Two days post-transfection, add KDalert Cell Lysis Buffer to the cells; incubate 20 min.
2. Add diluted Master Mix of assay reagents; incubate 4 min.
3. Read increase in fluorescence using a microplate or standard fluorometer or increase in absorbance with a spectrophotometer.