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

48 hours later, GAPDH mRNA levels were monitored by real-time RT-PCR (Figure 1A). For comparison, GAPDH siRNA was delivered by a standard transfection protocol (pre-plated) in two of the cell types (Figure 1B).

Figure 1. Efficiency of Reverse Transfection in Multiple Cell Types. (A) Indicated cell lines were plated in 96 well format (8000 cells/well) and simultaneously transfected by adding transfection complexes, prepared in Opti-mem serum-free medium (Life Technologies) by mixing 0.3 l siPORT NeoFX Transfection Reagent (Ambion) and 1 nM siRNA targeting GAPDH (Silencer GAPDH siRNA, Ambion) or non-silencing control (Silencer Negative Control #1 siRNA, Ambion). 48 hours post-transfection cells were harvested and analyzed by real-time RT-PCR for both target mRNA and 18S rRNA levels. Remaining gene expression was calculated as a percentage of target mRNA in cells transfected with siRNA targeting GAPDH and cells transfected with the non-silencing control siRNA. Data was normalized against the 18S rRNA signal. Transfections were performed in duplicate. Data are presented as means SD. (B) HepG2 and HeLa cells were both reverse transfected during plating and transfected after pre-plating the cells with the siRNA targeting GAPDH or negative control siRNA. 48 hours post-transfection, GAPDH expression was measured by real-time RT-PCR. Percent gene expression was calculated as GAPDH gene expression in GAPDH siRNA transfected cells compared to those transfected with the negative control siRNA.


As shown in Figure 1, the reverse transfection method provided significant reduction in GAPDH
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