( Materials and methods ...)Confirming gene silencing,,,mechanism by pGFP/GFP22 ,,,siRNA co-transfection

Materials and methods
HeLa S3 cells( ATCC, CCL-2.2) were plated at 5 x 104 cells /well in 24-well plates and incubated for 24 hours before transfection. Cells were transfected with 0.9 g of GFP22-siRNA (QIAGEN, Cat. # 1022064) per well using RNAiFect Reagent (QIAGEN, Cat. #301605). GFP22-siRNA has a homologous sequence to GFP mRNA. It was transfected unlabeled or labeled with Cy5. For GFP expression, cells were transfected with 0.2 g of pGFP in each well using Trans- Messenger Reagent (QIAGEN, Cat. # 301525). For co-transfection of GFP22-siRNA and pGFP, Trans- Messenger Reagent was also used. The recommended protocols, described in the QIAGEN RNAi- Fect handbook and the supplementary protocol for co-transfection of adherent cells with siRNA and plasmid DNA using TansMessenger Reagent was followed, using the optimized ratio of RNAi- Fect Reagent : siRNA and Trans- Messenger Reagent : siRNA of 1:5 as determined previously.
Analysis of GFP expression and Cy5 fluorescence was performed using the Agilent 2100 bioanalyzer with pressure cartridge for flow cytometric studies. For the analysis with the Agilent 2100 bioanalyzer, cells were trypsinized and washed twice in phosphate buffered saline (PBS), and then resuspended in Cell Buffer (CB) at a dilution of 1 x 106 -1.5 x 106 per milliliter. Cells were stained for 30 minutes at RT with the live dyes calcein-AM (Molecular Probes, Cat. # C-3099) or carboxynaphthofluorescein diacetate( CBNF) (Molecular Probes, Cat. # C-13196). Dye concentrations of 2.5 M were used for staining. Ten microliters of each sample were loaded on a microfluidic chip for analysis. Microfluidic chips and CB buffer were supplied with the Agilent Cell Fluorescence LabChip kit. Cy5 fluorescence indicated the efficiency of siRNA uptake and calcein-AM staining monitored the cell viabilit
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