for 1 min at 90°C, and then at 37°C for a further hour. The annealed siRNA can be aliquoted and stored in -20°C, but it is preferable to use it fresh.

5. Check annealed siRNA by 15% nondenaturing acrylamide gel electrophoresis (3.75 mL 40% acrylamide; 0.4 mL 2% bis; 1 mL 10X TBE; 100 pL 10% ammonium persulfate; 100 pL; 4.7 mL dH2O; and 10 pL TEMED).

6. As shown in Fig. 1, the sense and antisense oligonucleotides of E6 siRNA migrate faster and stain with less intensity with EtBr than with annealed E6 siRNA (Fig. 3).

Fig. 3. Migration of single-stranded E6 RNA sense and antisense oligonucleotides and annealed E6 siRNA on 15% nondenaturing acrylamide gel.

3.3. Human Cell Transfection With siRNA

3.3.1. Cell Line Maintenance

1 . CaSKi and SiHa epithelial cell lines are derived from human cervical carcinomas and contain the integrated HPV16 genome, about 600 copies (CaSKi) and one or two copies (SiHa). CaSKi cells also contain sequences related to HPV18 (ATCC CRL-1550). Both cell lines contain wild-type p53.

2. The cell lines need to be handled under Biosafety level 2 containment according to the ATCC.

3. Culture CaSKi cells in RPMI plus 10% FCS, 2 mM l-glutamine, and 1 mM sodium pyruvate.

4. Culture SiHa cells in MEM plus 10% FCS, 1 mM sodium pyruvate, and 0.1 mM nonessential amino acids.

5. Use human NDFs and human colon carcinoma epithelial HCT116 as HPV-negative cell line controls for this study.

6. Culture NDFs in MEM plus 15% FCS, 1.0 mM sodium pyruvate, and 0.1 mM nonessential amino acids.

7. Culture HCT116 cells in DMEM with 10% FCS.

8. Maintain all the cell lines with 100 U/mL penicillin and 100 pg/mL streptomycin at 37°C in 5% CO2 in air.

3.3.2. Cell Transfections

1. Trypsinize CaSKi and SiHa Cells and subculture them into 10-cm2 6-well plates without antibiotics, 1.5 x 105 cells per well.

2. Transfect the cells after 24 h with siRNA formulated into Oligofectamine Reagent (liposomes) according to the manufacturer's instructions.

3. For each well (about 2.5 x 105 cells), prepare the transfection mix as follows:

a. Dilute 10 pL 20 pM annealed siRNA with 175 pL Opti-MEM.

b. Then dilute 3 pL Oligofectamine with 12 pL Opti-MEM, and allow the mixture to sit for 5 min at room temperature.

c. Mix the diluted siRNA and Oligofectamine together, and allow it to sit for 20 min at room temperature before applying it to the cells as follows.

d. Wash the cells once with Opti-MEM, and then add 800 pL Opti-MEM to the well followed by 200 pL siRNA mixture.

e. After 4 h of incubation at 37°C in 5% CO2, add 0.5 mL normal growth media containing 30% FCS without antibiotics to give the final volume of 1.5 mL media per well.

4. Harvest the cells at different time points for analysis.

5. Note that the transfection could be expanded to a 10-cm dish (50 cm2) using the same cell/siRNA ratio, but the transfection efficiency may decrease.

3.3.3. Measurement of Transfection Efficiency

1. Transfection efficiency can be measured using FITC-dextran instead of siRNA.

2. Dissolve the FITC-dextran in PBS to 50 pg/pL, and centrifuge at 4000g for at least 60 min to get rid of free fluorescence using Centricon-10.

3. Dilute the centrifuged FITC-dextran in PBS at a final concentration of 20 pg/pL, and stored at 4°C.

4. For a 6-well plate (2.5 x 105 cells per well), use 10 pL of FITC-dextran per well instead of 10 pL siRNA to transfect CaSKi and SiHa.

5. To check the transfection efficiency, wash the transfected cells three times gently with PBS, and count the cells with internalized green fluorescence under the microscope, as shown in Fig. 4. For all the cell lines used in this study the transfection efficiencies are between 70 and 80%.

6. Confirm the transfection efficiencies using fluorescence-labeled siRNA (see Note 2).

0 0

Post a comment