Jacobs:Protocol siRNA transfection: Difference between revisions
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(New page: 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 siRN...) |
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siRNA transfection | siRNA transfection | ||
#ANKH siRNA transfection optimization ppt: [[Media:ANKH siRNA transfection optimize.pptx]] | |||
Part 1: Ratio Optimization (variable based on cell type) | Part 1: Ratio Optimization (variable based on cell type) | ||
Materials: | Materials: | ||
Lipfectamine 2000 | #Lipfectamine 2000 | ||
Opti-MEM I Reduced Serum Medium | #Opti-MEM I Reduced Serum Medium | ||
Stealth RNA/siRNA oligomers | #Stealth RNA/siRNA oligomers | ||
Stealth scramble siRNA | #Stealth scramble siRNA | ||
BLOCK-it Fluorescent Oligo (ex=494nm, em=519nm) (positive control for transfection reagent) | #BLOCK-it Fluorescent Oligo (ex=494nm, em=519nm) (positive control for transfection reagent) | ||
Fibronectin | #Fibronectin | ||
PBS (4C) | #PBS (4C) | ||
Glass/Plastic Bottom Dishes | #Glass/Plastic Bottom Dishes | ||
Coat glass with | #Coat glass with 1mL 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: | |||
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: | |||
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) | ||
Latest revision as of 14:29, 3 May 2010
siRNA transfection
- ANKH siRNA transfection optimization ppt: Media:ANKH siRNA transfection optimize.pptx
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 1mL 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
- 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)
