( y and trauma while ensuring ...)Delivering siRNAs to Difficult Cell Types

y and trauma while ensuring highly efficient siRNA delivery. In these experiments, electroporation was initially used to deliver Cy3-labeled siRNA targeting GAPDH into human primary mesenchymal stem cells (hMSC) and rat neuronal pheochromocytoma (PC-12) cells. 24 hours after electroporation almost every cell contained detectable amounts of Cy3-labeled siRNA (Figure 1). To confirm that the siRNAs were functional, GAPDH expression levels were measured in the hMSC cells (Figure 2). While this experiment resulted in efficient delivery of siRNA, it also resulted in low cell viability. Next we developed an electroporation buffer and protocol to significantly increase cell viability after electroporation.

Figure 1. Electroporation of Primary Cells and Hard-to-transfect Neuronal Cells.A Cy3-labeled GAPDH siRNA (1.5 g) was added to primary Human Mesenchymal Stem Cells (hMSC) or rat neuronal pheochromocytoma (PC-12) cells and electroporated (75 l) using hMSC-specific or PC-12-specific parameters. Cells were fixed 24 hours after electroporation, stained with DAPI (blue), and analyzed by fluorescence microscopy (Cy3-fluorescence; red).

Figure 2. Silencing Efficiency Depends on Amount of siRNA per Electroporation. Primary human mesenchymal stem cells (hMSC) were electroporated with various amounts of siRNA targeting GAPDH (0.1-3.0 g) or a scrambled negative control siRNA. 24 hours post-transfection, cells were harvested and analyzed by real-time RT-PCR for both GAPDH mRNA and 18S rRNA levels (Normalization control). mRNA remaining for GAPDH was calculated as a percentage of GAPDH mRNA detected in cells transfected with the negative control siRNA.
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