Jacobs:Protocol siRNA transfection: Difference between revisions
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#Coat glass with 400uL 1:100 Fibronectin:PBS before seeding | #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 | #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: | *For MC3T3s on glass slides in culture for multiple days past transfection: | ||
Start with: | *Start with: | ||
1 day: 325,000 cells/slide | *1 day: 325,000 cells/slide | ||
2 days: 150,000 cells/slide | *2 days: 150,000 cells/slide | ||
3 days: 80,000 cells/slide | *3 days: 80,000 cells/slide | ||
4 days: 50,000 cells/slide | *4 days: 50,000 cells/slide | ||
For MC3T3s on tissue culture dishes in culture for multiple days past transfection: | *For MC3T3s on tissue culture dishes in culture for multiple days past transfection: | ||
Start with: | *Start with: | ||
1 day: 400,000 cells/dish | *1 day: 400,000 cells/dish | ||
2 days: 200,000 cells/dish | *2 days: 200,000 cells/dish | ||
3 days: 100,000 cells/dish | *3 days: 100,000 cells/dish | ||
4 days: 50,000 cells/dish | *4 days: 50,000 cells/dish | ||
Follow: | *Follow: | ||
http://iccb.med.harvard.edu/screening/RNAi%20Libraries/Lipid%20Panel/16%20-%20Invitrogen%20Lipofectamine2000.pdf | 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 reagentspost-transfection incubation times (1 day, 2 days, 3 days, etc) | |||
*Take both brightfield and fluorescence imagescell siRNA uptake vs. cell death | |||
*use the best ratio | |||
Part 2: Verify Knock-down | 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) | *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) | *Western blot should include both negative and positive controls (untransfected cell protein lysate) |
Revision as of 08:59, 29 April 2010
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:
- 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 reagentspost-transfection incubation times (1 day, 2 days, 3 days, etc)
- Take both brightfield and fluorescence imagescell 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)