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High Throughput siRNA Delivery In Vitro: From Cell Lines to Primary Cells

n conditionsnamely electrical pulse length, voltage, and number of pulsesare different for different cell types (see Figure 4A and our siPORTer Conditions Chart).

Figure 4. Gene Silencing and Cell Viability After Electroporation with the siPORTer-96 Electroporation Chamber. Silencer GAPDH (Ambion) or Negative Control #1 siRNA (Ambion) (1.0 g) were electroporated using the siPORTer-96 (Ambion) and siPORT siRNA Electroporation Buffer (Ambion) and conditions listed in panel A into 8 cell types. Conditions listed were for electroporating eight identical samples at a time. (panel B): 24 h later, the cells were harvested and analyzed by real time RT-PCR for GAPDH mRNA levels. 18S rRNA levels were used to normalize GAPDH expression. Remaining gene expression was calculated as a percentage of GAPDH mRNA levels relative to levels obtained from cells electroporated with negative control siRNA.


Reproducibility of High Throughput Electroporation. To assess well-to-well reproducibility of the high throughput electroporation system, a GAPDH siRNA (48 samples) or negative control siRNA (48 samples) were simultaneously electroporated into NHDF-neo cells using the siPORTer-96 and siPORT siRNA Electroporation Buffer. Reduction in GAPDH levels was monitored 48 hours later by real-time PCR. As shown in Figure 5, GAPDH mRNA was reduced by 9296% (average reduction: 94.8%). In addition, cell viability remained above 85% across the plate. These results indicated that the siPORTer-96 provided highly reproducible results. Unlike other
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