( y. In cells transfected with pGFP GFP...)Confirming gene silencing,,,mechanism by pGFP/GFP22 ,,,siRNA co-transfection

y. In cells transfected with pGFP, GFP fluorescence indicated the transfection efficiency and the proportion of living cells was monitored by CBNF staining. The efficiency of gene silencing in cells co-transfected with Cy5-siRNA and pGFP was monitored by GFP fluorescence, and the viability of the cells was monitored by CBNF staining. Fluorescence microscopy was used to compare the 2100 bioanalyzer results 24 hours after transfection.
Results and discussion

Cells were analyzed 4, 8, and 24 hours after transfection using the 2100 bioanalyzer. As controls, untransfected cells were used, either unstained, stained with calcein- AM or CBNF alone. Controls were analyzed in parallel with the transfected cells (data not shown). In cells transfected with Cy5- labeled siRNA, Cy5 positive cells were detectable after 4 hours (figure 1). Transfection efficiency measurements after calcein staining showed that over 80 % of the live cells were Cy5-positive. This picture remained constant after 24 hours. In cells transfected with pGFP, the proportions of live cells expressing GFP increased from 22.4 % after 4 hours to 57.6 % after 24 hours. Co-transfection of pGFP and siRNA resulted in efficient gene silencing and results after each time point were compared and plotted (figure 2). A strong gene silencing effect was measured and by 24 hours the measured down-regulation of GFP expression was up to 77 %.

In a separate experiment, cells were transfected with pGFP alone, pGFP with unlabeled siRNA, and pGFP with Cy5-labeled siRNA to determine whether the Cy5 label affected gene silencing. Results, 24 hours after transfection, showed similar down regulation of the GFP expression, meaning that the Cy5 label did not interfere significantly with the gene silenci
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