Quantitative mitochondrial DNA copy number determination using droplet digital PCR with single-cell resolution

Area of Science:

• Cell Biology
• Genetics
• Biochemistry

Background:

• Mitochondria play crucial roles in cellular functions, with mitochondrial DNA (mtDNA) copy number varying across tissues, aging, and disease.
• mtDNA copy number alterations may serve as a biomarker for physiological changes within the human body.

Purpose of the Study:

• To develop a highly sensitive and quantitative method for measuring mitochondrial DNA copy number.
• To enable mtDNA copy number analysis in single cells and cell populations.
• To validate the method's ability to detect subtle changes in mtDNA copy number across diverse physiological contexts.

Main Methods:

• Development of droplet digital PCR (ddPCR) assay named droplet digital mitochondrial DNA measurement (ddMDM).
• ddMDM allows direct use of cell lysates without DNA purification or nuclear reference genes.
• Assay optimized for 96-well plates, generating data within 3 hours.

Main Results:

• ddMDM demonstrates high sensitivity, detecting mtDNA copy number differences indistinguishable by other methods.
• The assay successfully quantified mtDNA copy number changes in various physiological states.
• Quantitative changes were observed during cancer progression, cell cycle progression, T cell activation, and human aging.

Conclusions:

• The ddMDM method provides a sensitive and efficient tool for mtDNA copy number quantification.
• This technique enables the study of mtDNA dynamics in single cells and diverse physiological conditions.
• ddMDM facilitates the exploration of mtDNA copy number as a potential biomarker in health and disease.