The over-expression of short interfering RNA (siRNA) in vitro is widely used to study gene functions or to identify and validate new drug targets. We describe here a highly efficient method for selective gene suppression with siRNA in various established cell lines and primary cells (full protocol available on BioTechniques Protocols-On-Line). We also report the use of this technique for the rapid optimization of minimal siRNA concentrations which result in the highest % knockdown of mRNA and cell viability between 70–80%.
Current transfection techniques using lipids, viruses or specialty reagents maybe cytotoxic, labor intensive and costly. BTX Electroporation is a non-toxic, non-viral method which can be performed in a time efficient manner with inexpensive buffers. A variety of applications such as transfection of embryonic stem cells, in vivo, in ovo and in vitro gene transfer, cDNA and siRNA library screening, can all be efficiently performed by electroporation. BTX Harvard Apparatus offers the 96 Well High Throughput Electroporation System that boosts productivity, increases transfection efficiencies and delivers high cell viability. Results:
High-efficiency gene transfer and eliminated transfection-related toxicity was achieved for a variety of established cell lines as well as primary cells using BTX HT electroporation system (fig. 1).
Following the optimized BTX protocol, the rate of mRNA expression in the 96 well was directly proportional to the concentration of siRNA. The expression of mRNA in transfected cells ranged from 50% to 5% (fig. 2).
siRNA delivery can be accomplished rapidly and effectively using the BTX HT 96 Well System. In the above experiments we observed an 80 to 90 % inhibition of mRNA. The BTX system is easily used to optimize electroporation parameters for a variety of primary and established cell lines. BTX Electroporation Systems, optimized BTX protocols and low cost reagents deliver high transfection efficiency and high cell viability in all electroporation applications.
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