- Excellent viability
- Superior knockdown efficiency
- Equally successful with both adherent and suspension cells
- Optimal performance in several primary cells with a single reagent
- Easily establish optimized electroporation condition for different cell types
Electroporation with an effective buffer and optimized conditions could provide a highly efficient method for transfecting primary and hard-to-transfect cells with siRNAs. Ambion has developed an electroporation buffer that can be used with multiple types of electroporators to ensure highly efficient delivery of siRNA into cells while maintaining high cell viability.
Electroporation as an Alternative to Lipid-based Transfection
A successful siRNA experiment requires the appropriate transfection agent for your cells, and one that is optimized for siRNA delivery. Chemical transfection (e.g. using lipid based reagents) is being used routinely to deliver siRNAs into immortalized cells. Unfortunately, efficient transfer of siRNAs into primary cells by chemical transfection is restricted to a few cell types (1). Since primary cells are more similar to their in vivo counterparts than are immortalized cells, they serve as an important model system for in vivo applications. There is a need for better delivery methods for these cell types.
As an alternative to chemical transfection, Ambion has explored electroporation in primary and hard-to-transfect cell types. Electroporation involves applying an electric field pulse to induce the formation of microscopic pores in the cell membrane allowing molecules, ions, and water to traverse the membrane (2).
Ambion has developed a low-conductivity siRNA Electroporation Buffer that is designed to emulate the natural cytoplasmic composition of cells. This buffer works by facilitating rapid pore resealing, to ensure high cell viability after electroporation.
Optimization of Electroporation Conditions
Transfection via electroporation can be performed with two different commonly used electro-pulse generators: Gene Pulse Xcell (BioRad) and ECM 830 (BTX). Both electroporators performed approximately equivalently when the same electroporation conditions were applied, and resulted in the same siRNA uptake by several different cell types and similar reduction of gene expression (variability <10%).
Experiments have revealed that the following parameters can affect the optimal electroporation of siRNAs: electric field strength, pulse strength, and the number of field pulses. It is important to optimize these critical parameters for different cell types to ensure maximum delivery of siRNA by electroporation.
Ambion has optimized the electroporation conditions for several primary and hard-to-transfect cells, which can be seen in the table at right. Using the electroporation conditions as listed in the table, Ambion scientists have successfully knocked down p53 gene expression in normal human keratinocytes (Figure 1, below table). Further, both high levels of gene silencing and cell viability can be achieved in a broad spectrum of cells as seen in Figure 2. For more information on how to optimize your transfection conditions, please visit our siRNA Delivery Resource.
Each 1.5 ml tube of siPORT siRNA Electroporation Buffer is sufficient for 20 electroporations using standard 1 mm electroporation cuvettes (BioRad). The 20 ml size is particularly convenient for those using the siPORTer™-96 Electroporation Chamber and is sufficient for approximately 500 electroporations using the siPORTer-96.
A Complete System for Optimization of siRNA Delivery
In addition to siPORT siRNA Electroporation Buffer, Ambion offers the siPORT™ siRNA Electroporation Kit. This kit makes it easy to optimize electroporation conditions for any cell type. The siPORT siRNA Electroporation Kit contains the siRNA Electroporation Buffer, plus a Cy™3-labeled control siRNA, GAPDH control siRNA, and negative control siRNA. An instruction manual detailing electroporation optimization procedures is also included. The siRNA Electroporation Buffer contained in the kit is sufficient for performing 60 electroporations using standard 1 mm electroporation cuvettes (BioRad). The Cy3-labeled control siRNA makes it simple to monitor the uptake of siRNA into target cells by fluorescence microscopy. The GAPDH control siRNA can be used to optimize electroporation conditions in target cells, while the negative control siRNA can be used to confirm the specificity of the gene-specific siRNA and absence of non-specific toxic effects.
References
- Ovcharenko D (2003) Efficient delivery of siRNAs to human primary cells. Ambion TechNotes 10 (5): 15-16.
- Jarvis R, King A (2003) Optimizing chemical transfection and electroporation of siRNAs. Ambion TechNotes 10 (5): 12-15.
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