Protocol for performing PINK1 siRNA knockdown in mouse embryonic fibroblasts (MEFs)

For Primary MEFs seed 200,000 cells per well in a 6-well plate at a total volume of 2mL.

For Immortalised MEFs seed 100,000 cells per well in a 6-well plate at a total volume of 2mL.

Resuspend the dried siRNA – protocol for 5 nmol of purchased siRNA to reconstitute at [10micromolar (µM)].

Add 400µL of RNA-free water.

Add 100µL of 5x siRNA Buffer.

Incubate in hood for 0h 5m 0s, vortex vigorously and store at -20°C.

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.

Tube1 PINK1 siRNA:

Per well - 5µL of PINK1 siRNA at [10micromolar (µM)] diluted in 100µL OPTI-MEMTube 3

Tube 2:

Per well - 10µL of Lipofectamine diluted in 100µL of siRNA-OPTI-MEM

Tube 3 scramble siRNA

Per well - 5µL of scramble siRNA at [10micromolar (µM)] diluted in 100µL OPTI-MEM

Tube 4 (same as tube 2)

Per well - 10µL of Lipofectamine diluted in 100µL 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 0h 5m 0s at Room temperature.

Add drop by drop, 200µL of PINK1 siRNA mix to each well for PINK1 KD and similarly of the scramble siRNA for controls.

Make a 500x of Oligomycin/antimycin solution. The final concentration in the well is:

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

Add 4µL of this 500x solution will be to each well.

Treat cell with Oligomycin/antimycin A for 24h 0m 0s. OA should be added 48h 0m 0s after the addition of the siRNA. Include DMSO control.

Prepare working stock of MLi2 at a concentration of 100micromolar (µM) in DMSO.

Treat cells with MLi2(10nanomolar (nM)) for 1h 30m 0s (2 ul/well).

Lyse cells using 50µL of Lysis buffer/well

Lysate can be precleared by centrifugation 17000x g,0h 0m 0s for 0h 15m 0s at 4°C.

Perform protein estimation and subject lysate to immunoblotting.