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Delivering siRNAs to Difficult Cell Types


Electroporation Pulse Buffer and Electropulse Generators
Ambion's siPORT siRNA Electroporation Buffer is a low-conductivity buffer designed to emulate the natural composition of the cytoplasm. It enables delivery of siRNA into primary, neuronal, and other hard-to-transfect cell types, promoting siRNA-mediated knockdown of gene expression, while retaining high cell viability. Ambion's siPORT siRNA Electroporation Buffer works by facilitating rapid pore resealing. Using the siPORT siRNA Electroporation Buffer with square wave-type pulses, we were able to transfect hMSC cells, normal human dermal fibroblasts-neonatal (NHDF-Neo cells), normal human umbilical vein endothelial cells (HUVEC), Human acute T-cells (Jurkat) and rat neuronal PC-12 cells with high efficiencies (75.0-99.9%) and high cell viability (65-95%), as seen in Figure 3.

Figure 3. Silencing and Cell Viability after Electroporation. Successful gene silencing and high cell viability was achieved in 3 primary cell types: human mesenchymal stem cells (hMSC), normal human dermal fibroblasts-neonatal (NHDF-Neo), normal human umbilical vein endothelial cells (HUVEC) and 2 hard-to-transfect cell types: Jurkat and PC-12 (rat pheochromocytoma) cells. siRNA targeting GAPDH or scrambled negative control siRNA (1.5 g) were electroporated. 24 hours post-transfection, the cells were harvested and analyzed by real-time RT-PCR for target mRNA levels. 18S rRNA levels were used to normalize GAPDH expression. Remaining mRNA % was calculated as a percentage of mRNA compared with the PC-12 negative
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Antibodies were affinity purified using epitopes specific to XLF immobilized on solid support....
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