Jacobs:Protocol siRNA transfection

siRNA transfection

1. ANKH siRNA transfection optimization ppt: Media:ANKH siRNA transfection optimize.pptx‎

Part 1: Ratio Optimization (variable based on cell type)

Materials:

1. Lipfectamine 2000
2. Opti-MEM I Reduced Serum Medium
3. Stealth RNA/siRNA oligomers
4. Stealth scramble siRNA
5. BLOCK-it Fluorescent Oligo (ex=494nm, em=519nm) (positive control for transfection reagent)
6. Fibronectin
7. PBS (4C)
8. Glass/Plastic Bottom Dishes

1. Coat glass with 1mL 1:100 Fibronectin:PBS before seeding

*let sit for 1 hour before aspirating off extra

1. Plate cells 24 hours prior to transfection to reach ~50-60% confluency

*need to balance time in culture following transfection with confluency at transfection (don’t want cells to grow to overconfluency but cells will die if transfection reagents are strong) *transfection may slow cell division

      For MC3T3s on glass slides in culture for multiple days past transfection:

Start with: *1 day: 325,000 cells/slide *2 days: 150,000 cells/slide *3 days: 80,000 cells/slide *4 days: 50,000 cells/slide

       For MC3T3s on tissue culture dishes in culture for multiple days past transfection:

Start with: *1 day: 400,000 cells/dish *2 days: 200,000 cells/dish *3 days: 100,000 cells/dish *4 days: 50,000 cells/dish

Follow: http://iccb.med.harvard.edu/screening/RNAi%20Libraries/Lipid%20Panel/16%20-%20Invitrogen%20Lipofectamine2000.pdf

• use Opti-MEM for complex formation steps only
• may use antibiotic free 10%FBS-supplemented alpha-MEM media for first incubation
• remove reagents after 10 hours (8am-6pm or 10pm-8am is convenient)
• after removal of reagentspost-transfection incubation times (1 day, 2 days, 3 days, etc)

• Take both brightfield and fluorescence imagescell siRNA uptake vs. cell death

*use the best ratio

Part 2: Verify Knock-down

• Transfect a dish of the cells with the previously determined optimal seeding density and Lipo2000:siRNA ratio for different number of post-transfection incubations

*lyse the cells using RIPA buffer and run a BCA then Western blot to probe for the knockdown of your target protein. *make sure to use perform transfection in duplicate with negative (scramble siRNA)

       *Western blot should include both negative and positive controls (untransfected cell protein lysate)