( nealingof siRNA duplexes 5 nmol sin...)siRNA transfection optimization,,,with the Agilent 2100 bioanalyzer

nealing of siRNA duplexes, 5 nmol single stranded sequences targeting Lamin A/C (QIAGEN, Cat. #: 1022050), were incubated in 250 l siRNA Buffer (QIAGEN) for one min at 91 C followed by 1 h at 37 C. GFP siRNA (QIAGEN, GFP-22 siRNA, sense 3Cy5 modified sense and antisense 5-P(Phosphate)) was supplied annealed and ready to use in sterile buffer. Samples were then stored at 20 C until needed, and then incubated at 37 C for 20 min.
Electrophoretic measurement of purity and integrity
dsRNA electrophoresis analysis was performed using the DNA 1000 LabChip kit with the Agilent 2100 bioanalyzer. The kit includes dsDNA internal standards at 15 and 1500 bp for sample alignment (sizing) and quantitation. Lamin A/C siRNA was analyzed after 1:10 dilution in PBS.
Transfection and culture conditions
Lipid based TransMessenger and RNAiFect transfection reagents where kindly provided by QIAGEN GmbH. Human cervix carcinoma Hela S3 cells (DSMZ, ACC 161) were kept in culture with 10 % FBS in Hams F12 medium at 5 % CO2 atmosphere and 37 C. Twenty-four hours before transfection, 5x104 cells where seeded in 24 well plates. Transfection protocol was followed as described in QIAGENs manual for each transfection reagent, being the specific siRNA incubations times of 3 hours in serum free medium for Trans- Messenger and 17 hours in complete medium when RNAiFect was used. After transfection, cells were kept in culture medium until time of analysis, 24 hours after transfection was initiated.
Transfection efficiency measurements
Flow cytometry measurements were performed using the Cell Fluorescence LabChip kit with the Agilent 2100 bioanalyzer. After enzymatic cell detachment with Accutase (PAA laboratories, Cat. # L11-007) cells where resuspended
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