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

mRNA in all five cell types with as little as 1 nM siRNA. Interestingly, reverse transfection required approximately three-fold less siRNA to induce the same reduction in GAPDH mRNA expression as the corresponding cells transfected using the standard procedure (Figure 1). In addition, while standard pre-plated transfection was limited to transfecting 510 x 103 cells per well on a 96 well plate, reverse transfection exhibited similar reductions in target mRNA expression using 525 x 103 cells (data not shown).

Reproducibility of Reverse Transfection. To address reproducibility, we reverse transfected cells in eight wells of each of three different 96 well plates with 10 nM GAPDH siRNA. Two days later, we repeated the procedure with three more 96 well plates. Analysis of GAPDH mRNA 48 hours after transfection revealed that the reverse transfection procedure was indeed highly reproducible (Figure 2).

Figure 2. Reproducibility of Reverse Transfection Procedure. Silencer GAPDH (Ambion) and Negative Control siRNA #1 (Ambion) (10 nM) were reverse transfected in triplicate (Plates 13; 8 wells/plate) into HeLa cells in a 96 well format. 48 hours post-transfection, cells were harvested and analyzed by real-time RT-PCR for both target mRNA and 18S rRNA levels (Day 1). The entire experiment was repeated three days later (Day 3). Remaining gene expression was calculated as a percentage of target mRNA in cells transfected with siRNA targeting GAPDH compared to cells transfected with the negative control s
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