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Efficient Delivery of siRNAs to Human ,,, Primary Cells: Electroporation ,,, vs. Chemical Transfection

tend these results, validated siRNAs targeting CDK2, p53, and JAK1 were individually electroporated into the same set of primary cells. All three genes were efficiently silenced (Figure 4).

Figure 2. Electroporation of HUVEC Primary Cells. A Cy3-labeled GAPDH siRNA (1.33 g) was added to HUVEC primary cells and electroporated using the HUVEC-specific protocol (Amaxa) and Nucleofector machine (Amaxa). Cells were fixed 24 hours after electroporation and stained with DAPI (blue), Cy3 fluorescence (red).

Figure 3. Reduction of GAPDH Gene Expression in HUVEC, NHEK, and NHDF-neo Human Primary Cells. Different primary cell lines were electroporated using Amaxa Nucleofector technology. Varying amounts from 66 ng to 2.66 g of siRNA targeting GAPDH or 18S rRNA were used. 24 hours post-transfection, the cells were harvested and analyzed by real-time RT-PCR for both GAPDH mRNA and 18S rRNA levels. 18S rRNA levels were used to normalize the GAPDH data. Percent gene expression was calculated as a percentage of gene expression compared with the negative control siRNA. Duplicates were performed for each sample.

Figure 4. Validated siRNAs Elicit RNAi When Electroporated into Several Cell Types. Three siRNAs (1.33 g) targeting CDK2, p53, JAK1 and a scrambled sequence were electroporated into HUVEC, NHEK, NHDF-neo human primary cell lines. 24 hours post-transfection the cells were harvested and analyzed by real-time RT-PCR for gene expression levels. 18S rRNA levels were used to normalize the CDK2, p53, JAK1 data. Percent gene exp
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