Purification of cytosolic fraction and quantification of mtDNA by qPCR
Pietro De Camilli, Will Hancock-Cerutti, Zheng Wu, Gerald S. Shadel
Abstract
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.
Attachments
dxwwbkjz7.pdf
Steps
Cell culture and purification of cytosolic fraction
Plate HeLa cells in DMEM 15cm plates (3.5 x 106 cells per plate).
The following day, prepare cytosolic buffer with fresh digitonin.
Prepare lysis buffer.
Trypsinize cells and centrifuge at 1500rpm,0h 0m 0s for 0h 5m 0s at22°C.
Resuspend cells in PBS and count cells.
Collect the same number of cells from each genotype (5 x 106) and centrifuge at 1500rpm,0h 0m 0s for 0h 5m 0s at 22°C.
Resuspend cells in 1mL PBS and transfer 50µL to a prechilled Eppendorf another (WCE). Keep On ice.
Transfer the remaining 950µL to a prechilled Eppendorf and centrifuge at 4500rpm,0h 0m 0s for 0h 5m 0s at 22°C.
Remove supernatant and resuspend pellet in 500µL cytosolic buffer.
Rotate for 0h 10m 0s at 4°C.
Centrifuge extract at 980x g,0h 0m 0s for 0h 3m 0s at 4°C. Transfer supernatant to new Eppendorf tube and save pellet for analysis (Pel).
Centrifuge supernatant at 17000x g,0h 0m 0s for 0h 10m 0s at 4°C.
Collect supernatant (Cyt).
Purify DNA from WCE and Cyt fractions using DNeasy Kit (Qiagen).
Measure DNA concentration and dilute samples 1:10.
qPCR
Combine 10µL SYBR Green Master Mix (BioRad) with 6.78µL Sterile Water (American Bio) per sample.
Combine 16.78µL diluted SYBR Green Master Mix with 0.61µL each of 10micromolar (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.
Pipette 2µL of diluted DNA from step 15 in well with SYBR Green Master Mix.
Cover plate with Optical Adhesive Covers (Applied Biosystems).
Spin down plate in table top centrifuge.
Run qPCR in CFX96 Real-Time System (BioRad) using the following protocol:
| A | B | C |
|---|---|---|
| 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 |
Data analysis
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.
Subtract the ΔCt of the control sample from each sample ΔCt to calculate the ΔΔCt value.
Calculate relative expression using the 2−ΔΔCt method.
WT mtDNA abundance was given a value of 1.
