TITLE: Purification of cytosolic fraction and quantification of mtDNA by qPCR
AUTHORS: Will Hancock-Cerutti, Zheng Wu, Gerald S. Shadel, Pietro De Camilli

[DESCRIPTION]
This protocol describes the purification of a cytosolic fraction depleted of membrane from cultured cells, and the quantification of mitochondrial DNA (mtDNA) in this fraction by qPCR.

[STEPS]
SECTION: Cell culture and purification of cytosolic fraction
1. Plate HeLa cells in DMEM 15 cm plates (3.5 x 106 cells per plate).

SECTION: Cell culture and purification of cytosolic fraction
2. The following day, prepare cytosolic buffer with fresh digitonin.
SECTION: Cell culture and purification of cytosolic fraction
3. Prepare lysis buffer.
SECTION: Cell culture and purification of cytosolic fraction
4. Trypsinize cells and centrifuge at 1500 rpm for 5 min at22 °C.

SECTION: Cell culture and purification of cytosolic fraction
5. Resuspend cells in PBS and count cells.
SECTION: Cell culture and purification of cytosolic fraction
6. Collect the same number of cells from each genotype (5 x 106) and centrifuge at 1500 rpm for 5 min at 22 °C.

SECTION: Cell culture and purification of cytosolic fraction
7. Resuspend cells in 1 mL PBS and transfer 50 µL to a prechilled Eppendorf another (WCE). Keep on ice.

SECTION: Cell culture and purification of cytosolic fraction
8. Transfer the remaining 950 µL to a prechilled Eppendorf and centrifuge at 4500 rpm for 5 min at 22 °C.

SECTION: Cell culture and purification of cytosolic fraction
9. Remove supernatant and resuspend pellet in 500 µL cytosolic buffer.

SECTION: Cell culture and purification of cytosolic fraction
10. Rotate for 10 min at 4 °C.

SECTION: Cell culture and purification of cytosolic fraction
11. Centrifuge extract at 980 x g for 3 min at 4 °C. Transfer supernatant to new Eppendorf tube and save pellet for analysis (Pel).

SECTION: Cell culture and purification of cytosolic fraction
12. Centrifuge supernatant at 17000 x g for 10 min at 4 °C.

SECTION: Cell culture and purification of cytosolic fraction
13. Collect supernatant (Cyt).
SECTION: Cell culture and purification of cytosolic fraction
14. Purify DNA from WCE and Cyt fractions using DNeasy Kit (Qiagen).
SECTION: Cell culture and purification of cytosolic fraction
15. Measure DNA concentration and dilute samples 1:10.
SECTION: qPCR
16. Combine 10 µL SYBR Green Master Mix (BioRad) with 6.78 µL Sterile Water (American Bio) per sample.

SECTION: qPCR
17. Combine 16.78 µL diluted SYBR Green Master Mix with 0.61 µL each of 10 Micromolar (µM) forward and reverse primers per sample. Pipette this mixture into wells of 96-well qPCR plate. Perform at least two technical replicates for each sample.

SECTION: qPCR
18. Pipette 2 µL of diluted DNA from step 15 in well with SYBR Green Master Mix.

SECTION: qPCR
19. Cover plate with Optical Adhesive Covers (Applied Biosystems).
SECTION: qPCR
20. Spin down plate in table top centrifuge.
SECTION: qPCR
21. Run qPCR in CFX96 Real-Time System (BioRad) using the following protocol:
 
          95 ˚C   3 min      95 ˚C   10 sec   Repeat 39x    55 ˚C   10 sec      72˚C   30 sec      95 ˚C   10 sec      65 ˚C   5 sec      95 ˚C   5 sec    
 
SECTION: Data analysis
22. Subtract the nuclear gene (hB2M) mean threshhold cycle (Ct) values from WCE samples from mtDNA amplicon of interest mean Ct values from Cyt samples to calculate ΔCt.
SECTION: Data analysis
23. Subtract the ΔCt of the control sample from each sample ΔCt to calculate the ΔΔCt value.
SECTION: Data analysis
24. Calculate relative expression using the 2−ΔΔCt method.
SECTION: Data analysis
25. WT mtDNA abundance was given a value of 1.