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Delivering siRNAs to Difficult Cell Types

control siRNA.


Electroporators
Transfection using the siPORT siRNA Electroporation Buffer was performed with two different electro-pulse generators: Gene Pulser Xcell (Bio-Rad) and ECM 830 (BTX). Both electroporators performed approximately equivalently when the same electroporation parameters were used, resulting in equivalent levels of siRNA uptake by several different cell types and similar reduction of gene expression (variability <10%; data not shown). The Gene Pulser Xcell System performed very consistently without sample-to-sample electropulse fluctuations across multiple experiments. The ECM 830 electropulse generator instrument performed with 5-10% sample-to-sample electropulse fluctuations across multiple experiments.


Optimizing siRNA Electroporation Parameters
Experiments revealed that different primary cell types require different electroporation parameters. For example, in a standard 1 mm electroporation cuvette, HUVEC cells require 1 electropulse, 150 S, and 250 Volts; whereas primary NHDF-Neo cells require 2 pulses, 70 S, and 900 Volts. Therefore, as with any transfection, it is important to optimize some critical protocol parameters to ensure maximum delivery of siRNA by electroporation. Transfection efficiency is largely affected by the concentration of siRNA, and can be modulated by varying the siRNA amount within a limited range. To demonstrate the relationship between
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