Apr 18, 2022

Public workspacePurification of cytosolic fraction and quantification of mtDNA by qPCR

DOI
dx.doi.org/10.17504/protocols.io.14egnz12yg5d/v1
  • Will Hancock-Cerutti1,2,3,
  • Zheng Wu4,5,
  • Gerald S. Shadel5,
  • Pietro De Camilli1,3
  • 1Departments of Neuroscience and of Cell Biology, Howard Hughes Medical Institute, Program in Cellular Neuroscience, Neurodegeneration and Repair, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
  • 2Interdisciplinary Neuroscience Program and MD-PhD Program, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
  • 3Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815;
  • 4Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
  • 5Salk Institute for Biological Studies, La Jolla, CA, USA
William Hancock-Cerutti
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DOI: dx.doi.org/10.17504/protocols.io.14egnz12yg5d/v1
Protocol Citation: Will Hancock-Cerutti, Zheng Wu, Gerald S. Shadel, Pietro De Camilli 2022. Purification of cytosolic fraction and quantification of mtDNA by qPCR. protocols.io https://dx.doi.org/10.17504/protocols.io.14egnz12yg5d/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: July 08, 2021
Last Modified: May 31, 2024
Protocol Integer ID: 51399
Keywords: Mitochondria DNA (mtDNA), Cytosol fractionation, qPCR, ASAPCRN
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.
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Materials
Solutions to prepare:

DMEM solution:
AB
FBS10%
Penicillin100 U/ml
Streptomycin100 mg/ml
L-glutamine2 mM
Cytoplasmic buffer:
AB
NaCl150 mM
HEPES50 mM
Digitonin, pH 7.41 mg/mL
Lysis buffer:
AB
NaCl150 mM
HEPES50 mM
Digitonin, pH 7.41 mg/mL
SDS supplemented with Protease Inhibitor Cocktail (Roche)1%
Cell culture and purification of cytosolic fraction
Cell culture and purification of cytosolic fraction
Plate HeLa cells in DMEM Amount15 cm 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 Centrifigation1500 rpm for Duration00:05:00 atTemperature22 °C .

5m
Centrifigation
Resuspend cells in PBS and count cells.
Collect the same number of cells from each genotype (5 x 106) and centrifuge at Centrifigation1500 rpm for Duration00:05:00 at Temperature22 °C .

5m
Centrifigation
Resuspend cells in Amount1 mL PBS and transfer Amount50 µL to a prechilled Eppendorf another (WCE). Keep TemperatureOn ice .

Transfer the remaining Amount950 µL to a prechilled Eppendorf and centrifuge at Centrifigation4500 rpm for Duration00:05:00 at Temperature22 °C .

5m
Centrifigation
Remove supernatant and resuspend pellet in Amount500 µL cytosolic buffer.

Rotate for Duration00:10:00 at Temperature4 °C .

10m
Centrifuge extract at Centrifigation980 x g for Duration00:03:00 at Temperature4 °C . Transfer supernatant to new Eppendorf tube and save pellet for analysis (Pel).

3m
Centrifigation
Centrifuge supernatant at Centrifigation17000 x g for Duration00:10:00 at Temperature4 °C .

10m
Centrifigation
Collect supernatant (Cyt).
Purify DNA from WCE and Cyt fractions using DNeasy Kit (Qiagen).
Measure DNA concentration and dilute samples 1:10.
qPCR
qPCR
Combine Amount10 µL SYBR Green Master Mix (BioRad) with Amount6.78 µL Sterile Water (American Bio) per sample.

Combine Amount16.78 µL diluted SYBR Green Master Mix with Amount0.61 µL each of Concentration10 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.

Pipetting
Mix
Pipette Amount2 µL of diluted DNA from step 15 in well with SYBR Green Master Mix.

Pipetting
Cover plate with Optical Adhesive Covers (Applied Biosystems).
Spin down plate in table top centrifuge.
Centrifigation
Run qPCR in CFX96 Real-Time System (BioRad) using the following protocol:
ABC
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
PCR
Data analysis
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.