TITLE: Protocol for performing PINK1 siRNA knockdown in mouse embryonic fibroblasts (MEFs)
AUTHORS: Enrico Bagnoli, Miratul Muqit

[DESCRIPTION]
This protocol details the siRNA knockdown in mouse embryonic fibroblasts (MEFs) for PINK1, but applicable for any other target.

[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.

[STEPS]
SECTION: Day 1 - Cell Seeding and siRNA preparation
1. For Primary MEFs seed 200,000 cells per well in a 6-well plate at a total volume of 2 mL.



SECTION: Day 1 - Cell Seeding and siRNA preparation
4.1. Add 400 µL of RNA-free water.

SECTION: Day 1 - Cell Seeding and siRNA preparation
4.3. Incubate in hood for 5 min, vortex vigorously and store at -20 °C.

 

SECTION: Day 2 - siRNA Knock-Down
5. 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.

 

SECTION: Day 2 - siRNA Knock-Down
5.2. Tube 2:
Per well - 10 µL of Lipofectamine diluted in 100 µL of siRNA-OPTI-MEM

SECTION: Day 2 - siRNA Knock-Down
5.3. Tube 3 scramble siRNA
Per well - 5 µL of scramble siRNA at [10 micromolar (µM)] diluted in 100 µL OPTI-MEM

SECTION: Day 2 - siRNA Knock-Down
5.4. Tube 4 (same as tube 2)
Per well - 10 µL of Lipofectamine diluted in 100 µL of siRNA-OPTI-MEM

SECTION: Day 1 - Cell Seeding and siRNA preparation
2.  For Immortalised MEFs seed 100,000 cells per well in a 6-well plate at a total volume of 2 mL.

SECTION: Day 1 - Cell Seeding and siRNA preparation
4. Resuspend the dried siRNA – protocol for 5 nmol of purchased siRNA to reconstitute  at                    [10 micromolar (µM)].

SECTION: Day 1 - Cell Seeding and siRNA preparation
4.2. Add 100 µL of 5x siRNA Buffer.
SECTION: Day 2 - siRNA Knock-Down
5.1. Tube1 PINK1 siRNA: 
Per well - 5 µL of PINK1 siRNA at [10 micromolar (µM)] diluted in 100 µL OPTI-MEMTube 3

SECTION: Day 2 - siRNA Knock-Down
6. Vortex slowly and combine the content of Tube 1 with tube 2 (PINK1 siRNA) and similarly with tube 3 and 4.
SECTION: Day 2 - siRNA Knock-Down
7. Vortex again slowly and incubate for 5 min at Room temperature.

SECTION: Day 2 - siRNA Knock-Down
8. Add drop by drop, 200 µL of PINK1 siRNA mix to each well for PINK1 KD and similarly of the scramble  siRNA for controls.

 

SECTION: Day 4 - Mitochondrial depolarization
9. Make a 500x of Oligomycin/antimycin solution. The final concentration in the well is:

oligo: 1 micromolar (µM)
antimycin: 10 micromolar (µM)

SECTION: Day 4 - Mitochondrial depolarization
10. Treat cell with Oligomycin/antimycin A for 1440 min. OA should be added 2880 min after the addition of the siRNA. Include DMSO control.

SECTION: Day 4 - Mitochondrial depolarization
9.1. Add 4 µL of this 500x solution will be to each well.


 



SECTION: Day 5 - Cell lysis
11. Prepare working stock of MLi2 at a concentration of 100 micromolar (µM) in DMSO.

SECTION: Day 5 - Cell lysis
12. Treat cells with MLi2(10 nanomolar (nM)) for 90 min (2 ul/well).

SECTION: Day 5 - Cell lysis
13. Lyse cells using 50 µL of Lysis buffer/well


SECTION: Day 5 - Cell lysis
14. Lysate can be precleared by centrifugation 17000 x g for 15 min at 4 °C.

SECTION: Day 5 - Cell lysis
15. Perform protein estimation and subject lysate to immunoblotting.
3.