May 13, 2024
Protocol for performing PINK1 siRNA knockdown in mouse embryonic fibroblasts (MEFs)
- Enrico Bagnoli1,
- Miratul Muqit1
- 1Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK
Protocol Citation: Enrico Bagnoli, Miratul Muqit 2024. Protocol for performing PINK1 siRNA knockdown in mouse embryonic fibroblasts (MEFs). protocols.io https://dx.doi.org/10.17504/protocols.io.kxygx343zg8j/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 13, 2023
Last Modified: May 13, 2024
Protocol Integer ID: 92308
Keywords: ASAPCRN
Funders Acknowledgement:
ASAP
Grant ID: ASAP-000463
Abstract
This protocol details the siRNA knockdown in mouse embryonic fibroblasts (MEFs) for PINK1, but applicable for any other target.
Attachments
926-2384.docx
17KB
Guidelines
72-Hr siRNA knockdown in MEFs in a 6-well plate
If using Immortalised MEFs – seed 100,000 cells per well.
If using Primary MEFs – seed 200,000 cells per well.
Materials
Key Reagents
siRNA as purchased from Horizon Discovery as ON-TARGETplus siRNA Reagents (Dharmacon‱ siRNA solutions).
Note
Protocol for 5 nmol quantities of purchased siRNA
Lipofectamine™ RNAiMAX Transfection ReagentThermo FisherCatalog #13778150
Oligomycin
Antimycin
MLi2
Day 1 - Cell Seeding and siRNA preparation
Day 1 - Cell Seeding and siRNA preparation
For Primary MEFs seed 200,000 cells per well in a 6-well plate at a total volume of 2 mL .
For Immortalised MEFs seed 100,000 cells per well in a 6-well plate at a total volume of 2 mL .
Day 1 - Cell Seeding and siRNA preparation
Day 1 - Cell Seeding and siRNA preparation
Resuspend the dried siRNA – protocol for 5 nmol of purchased siRNA to reconstitute at [10 micromolar (µM) ].
Add 400 µL of RNA-free water.
Add 100 µL of 5x siRNA Buffer.
Incubate in hood for 00:05:00 , vortex vigorously and store at -20 °C .
Note
The protocol can be amended if the nmol quantity of siRNA purchased is higher/lower by
adjusting the resuspension to ensure a final [10 micromolar (µM) ] concentration.
5m
Day 2 - siRNA Knock-Down
Day 2 - siRNA Knock-Down
5m
Prepare mastermix according to the number of wells. Four tubes have to be prepared, 2 with Optimem and lipofectamine and one each with the PINK1 and scramble siRNA respectively.
Note
For PINK1 knockdown a final concentration of 25 nM of siRNA in each well is used.
Tube1 PINK1 siRNA:
Per well - 5 µL of PINK1 siRNA at [10 micromolar (µM) ] diluted in 100 µL OPTI-MEMTube 3
Tube 2:
Per well - 10 µL of Lipofectamine diluted in 100 µL of siRNA-OPTI-MEM
Tube 3 scramble siRNA
Per well - 5 µL of scramble siRNA at [10 micromolar (µM) ] diluted in 100 µL OPTI-MEM
Tube 4 (same as tube 2)
Per well - 10 µL of Lipofectamine diluted in 100 µL of siRNA-OPTI-MEM
Vortex slowly and combine the content of Tube 1 with tube 2 (PINK1 siRNA) and similarly with tube 3 and 4.
Vortex again slowly and incubate for 00:05:00 at Room temperature .
5m
Add drop by drop, 200 µL of PINK1 siRNA mix to each well for PINK1 KD and similarly of the scramble siRNA for controls.
Note
For an experiment with 4x 6 well plates (WT and mutant cells treated with scramble or PINK1
the following volumes can be used:
- Tube1 and tube 3: 60 µL siRNA + 1200 µL OPtiMEM
- Tube 2 and 4: 120 µL of lipofectamine + 1200 µL of otpimem
- This gives 2580 µL of each mix (180 µL spare)
Day 4 - Mitochondrial depolarization
Day 4 - Mitochondrial depolarization
5m
Make a 500x of Oligomycin/antimycin solution. The final concentration in the well is:
- oligo: 1 micromolar (µM)
- antimycin: 10 micromolar (µM)
Add 4 µL of this 500x solution will be to each well.
Note
For a 100 µL of 500x solution, we use:
| A | B | |
| Compound | Quantity | |
| Antimycin A 50 mM solution | 10 ul | |
| Oligomycin 6.4 mM solution | 7.8 ul | |
| DMSO | 82.2 ul |
Treat cell with Oligomycin/antimycin A for 24:00:00 . OA should be added 48:00:00 after the addition of the siRNA. Include DMSO control.
3d
Day 5 - Cell lysis
Day 5 - Cell lysis
1h 50m
Prepare working stock of MLi2 at a concentration of 100 micromolar (µM) in DMSO.
Treat cells with MLi2(10 nanomolar (nM) ) for 01:30:00 (2 ul/well).
1h 30m
Lyse cells using 50 µL of Lysis buffer/well
Lysate can be precleared by centrifugation 17000 x g for 00:15:00 at 4 °C .
15m
Perform protein estimation and subject lysate to immunoblotting.