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

The KDalert assay is an integral part of Ambion’s suite of products to optimize siRNA transfection into cells. Of particular note are the Silencer® CellReady™ siRNA Transfection Optimization Kit and the Silencer® siRNA Transfection II Kit, which can be  used to deliver GAPDH and negative control siRNAs into cells under a range of transfection conditions. The KDalert Kit can then rapidly identify transfection conditions that maximize silencing while minimizing transfection-associated cytotoxicity. When used together, these kits enable researchers to rapidly determine optimal transfection conditions, which can then be used for all subsequent transfections into that cell type. (The Silencer® siRNA Starter Kit, which combines these two products for researchers new to RNAi, is also available).

Rapid Measurment of Silencing and Cell Viability

To demonstrate the ease of siRNA delivery optimization afforded by combining the Silencer CellReady siRNA Transfection Optimization Kit and the KDalert GAPDH Assay Kit, we used the two kits to optimize siRNA delivery into HepG2 cells, a cell type that can be difficult to transfect. We created a matrix of transfection conditions in a 96 well Silencer CellReady siRNA Transfection Optimization Plate in which cell number and the amount of siPORT™ NeoFX™ Transfection Agent (included in the kit) were varied. HepG2 cells were reverse transfected with either Silencer GAPDH siRNA or Silencer Negative Control #1 siRNA; these siRNAs are pre-aliquotted into plates as part of the Silencer CellReady siRNA Transfection Optimization Kit. Two days after transfection, the medium was removed and the cells were lysed using the reagents and protocol in the KDalert Kit. GAPDH activity in each of the lysates was then determined using the KDalert assay.

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

The optimal transfection conditions for HepG2 cells were used to transfect Ambion Silencer® Pre-designed siRNAs targeting four additional genes (CDK2, STAT1, JAK1, and PCNA). Two days after transfection the amount of gene knockdown was measured for each transfected HepG2 sample using real-time qRT-PCR (Figure 2). Each gene target was efficiently silenced using the optimized transfection procedure. For these genes, the optimized transfection conditions provided the highest levels of target silencing compared with other tested transfection optimization conditions (data not shown).

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

The KDalert assay is an excellent complement to Ambion’s suite of control siRNAs and reagents for siRNA transfection. The great ease and speed of the KDalert procedure make it an ideal replacement for qRT-PCR based methods for transfection optimization.

Scientific Contributors

Luis Foncerrada, Kevin Kelnar • Ambion, Inc.