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

*let sit for 1 hour before aspirating off extra *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
 * 1) Coat glass with 1mL 1:100 Fibronectin:PBS before seeding
 * 1) Plate cells 24 hours prior to transfection to reach ~50-60% confluency

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)

*use the best ratio
 * Take both brightfield and fluorescence imagescell siRNA uptake vs. cell death

Part 2: Verify Knock-down

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