Electroporation: An Advantageous Alternative to Chemical Transfection
Human primary cells are often preferred for RNAi experiments because they are more similar to their in vivo counterparts than are immortalized cells. Although common chemical transfection methods are effective in many transformed cell lines, they are often not effective for primary cells . For these cell types as well as suspension and other hard-to-transfect cells, electroporation is a valuable alternative.
Electroporation involves applying an electric field pulse to induce the formation of microscopic pores in the cell membrane. These pores allow nucleic acids such as siRNA to traverse the membrane. Under specific pulse conditions, the pores reseal, and the electroporated cells recover and resume growth.
The siPORTer-96 Electroporation Chamber (patent pending; Figure 1) works with the Bio-Rad Gene Pulser Xcell pulse generator to allow high throughput siRNA electroporation. The specially designed electrodes enable rapid electroporation without requiring expensive, consumable electroplates or cuvettes. When you use the siPORTer-96 Electroporation Chamber to deliver siRNA, you will be able to detect RNAi-induced gene silencing just a few hours after delivery  (also, see Deliver siRNAs Into Primary Cells).
Figure 1. siPORTer-96 Electroporation Chamber. This photograph shows the electroporation chamber base with the electrode cartridge. The siPORTer-96 Electroporation Chamber was designed for safety and convenience. The assembled electrode cartridge protects samples from contamination--samples can be loaded inside a laminar flow hood, and the assembled electrode cartridge can be carried to the electroporation chamber base. Alternatively, the entire device can be set up inside a laminar flow hood.
Greater Cell Viability
Most existing electroporation protocols were developed to deliver plasmid DNA into cell nuclei. These protocols often result in high rates of cell mortality. Since siRNAs need only be delivered into the cytoplasm, milder electroporation conditions can be used. Using milder electroporation conditions and siPORT siRNA Electroporation Buffer (included) minimizes cellular mortality and trauma without sacrificing efficient siRNA delivery . With electroporation, transfection occurs immediately, and cells can be transfected regardless of their cell cycle stage.
High Throughput, Reproducible Results
A multiwell electroporator makes optimizing electroporation conditions for new cell types much easier than single well electroporators. In addition, high throughput electroporation of siRNA libraries into primary cells is an extremely effective research tool for investigating gene function across entire gene classes. The siPORTer-96 Electroporation Chamber allows screening of large numbers of siRNAs rapidly, reproducibly, and in parallel. Using the same pulse conditions in a single run, other parameters can be identical or varied across wells. The standard sample volume is just under 50 l, and as few as 2.5 x 104 cells can be used per reaction. This means that ~100 siRNAs could be screened using only 2.5 x 106 cells and 4.4 ml of siPORT siRNA Electroporation Buffer.
The siPORTer-96 Electroporation Chamber yields results that are remarkably reproducible (Figure 2). 96 samples were electroporated using identical conditions. Half of the samples received an siRNA targeting GAPDH (Silencer GAPDH siRNA, Cat# 4605), and half received a scrambled Negative Control siRNA (Silencer Negative C ontrol #1 siRNA, Cat# 4611). Data are shown for the GAPDH siRNA-treated samples. Normal expression levels of GAPDH were reduced by a mean of 94.8% with a coefficient of variance of 18.9%. Variability is attributed to steps in the procedure such as pipetting error, measurement error, and assay error, as well as true biological variability.
Figure 2. Reduction of GAPDH Gene Expression in NHDF-neo Cells. siRNA (1 g) was electroporated into NHDF-neo (normal numan dermal fibroblast cells, an adherent primary cell type) using the siPORTer-96 Electroporation Chamber. Samples 1-48 were transfected with an siRNA targeting GAPDH (Silencer GAPDH siRNA, this figure), and samples 49-96 were transfected with a negative control siRNA (Silencer Negative Control #1 siRNA, data not shown). 48 hours after transfection, the cells were harvested and analyzed by real-time RT-PCR for GAPDH expression levels. 18S rRNA levels were used to normalize GAPDH expression. Remaining Gene Expression was calculated as a percentage of GAPDH gene expression compared with the averaged value from cells transfected with the negative control siRNA.
Figure 3 shows results of experiments used to identify optimal electroporation conditions for NHDF-neo primary cells. An siRNA targeting GAPDH and a Negative Control siRNA were again used for this experiment. Both remaining GAPDH expression levels and cell viability were measured. These data illustrate the impressive gene silencing that can be achieved by delivering a potent siRNA with the siPORTer-96 Electroporation Chamber while maintaining cell viability. A wide range of parameters are effective in delivering siRNA to adherent cells like NHDF-neo cells.
Figure 3. GAPDH Gene Silencing and Cell Viability Under Various Electroporation Conditions. siRNA (1 g) targeting GAPDH (Silencer GAPDH siRNA, Ambion) or a Negative Control siRNA (Silencer Negative Control #1 siRNA, Ambion) were electroporated into normal human dermal fibroblast cells (an adherent primary cell type) using the siPORTer-96 Electroporation Chamber. Four samples were electroporated at a time, either with an siRNA targeting GAPDH (two samples) or with the negative control siRNA (two samples). The voltage was varied between 200 and 600 V for either 250 or 400 sec as labeled. 24 hours after electroporation, the cells were harvested and analyzed by real-time RT-PCR for GAPDH expression levels. 18S rRNA levels were used to normalize GAPDH expression. Remaining Gene Expression was calculated as a percentage of GAPDH gene expression compared with samples transfected with the negative control siRNA (bars). Cell viability was measured with ViaCount Cell Viability assay (Guava) (diamonds).
When used with Bio-Rad's Gene Pulser Xcell pulse generator, Ambion's siPORTer-96 Electroporation Chamber enables efficient high throughput electroporation of siRNA. Coupled with the siPORT siRNA Electroporation Buffer, the electroporator provides highly efficient siRNA delivery into primary and other hard-to-transfect cell types with minimal cell mortality. Standard sample volume is 44 l, and enough siPORT siRNA Electroporation Buffer (12 X 1.5 ml) is supplied with the siPORTer-96 Elec troporation Chamber for 360 samples. Conditions can be identical or varied across samples, and multiple wells allow for faster condition optimization. There is no need for expensive, disposable electrode plates or cuvettes. The unit is simply wiped with ethanol to clean.
Dmitriy Ovcharenko, Rich Jarvis, Po-Tsan Ku, Kevin Kelnar, Vince Pallotta, Scott Hunicke-Smith Ambion, Inc.