Jacobs:Protocol siRNA transfection

siRNA transfection

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

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

Coat glass with 400uL 1:100 Fibronectin:PBS before seeding -let sit for 1 hour before aspirating off extra 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)