Mitochondrial DNA (mtDNA) released into the cytosol, also known as mtDNA leakage, is a well-established damage-associated molecular pattern (DAMP) that triggers innate immune activation, inflammation, and cell death. Once in the cytosol, mtDNA can potently activate the cyclic GMP-AMP synthase–stimulator of interferon genes (cGAS-STING) signaling pathway, linking mitochondrial stress to broader inflammatory responses. Along with changes in mtDNA copy number, integrity, and methylation status, cytosolic mtDNA leakage represents a critical indicator of mitochondrial dysfunction.

The RayBio^® Human Mitochondrial DNA Leakage Relative Quantification Kit offers a fast, reliable, and quantitative solution for measuring mtDNA leakage in the cytosol of human cells. The kit employs a TaqMan™ probe-based qPCR assay to accurately quantify both genomic DNA and mtDNA fragments across subcellular compartments - including whole cells, isolated mitochondria, and cytosolic fractions. Compatible with a broad range of primary cells and established cell lines, this kit enables the study of mtDNA leakage across diverse genetic, biological, and pathophysiological contexts.

• Pipette tips (barrier tips recommended) and microcentrifuge tubes
• PBS
• Microcentrifuge
• Magnetic separator for microcentrifuge tubes (2 mL)
• Vortex mixer
• Tissue homogenizer if using tissue samples (Dounce tissue grinder recommended)
• Refrigerated centrifuge with swing out rotor and refrigerated microcentrifuge
• Compatible PCR Plate or PCR tubes
• Fluorescence qPCR instrument capable of reading FAM (494 nm maximum absorption, 518 nm maximum emission) and VIC (532 nm maximum absorption, 552 nm maximum excitation) channels

1. Prepare all reagents, samples and standards as instructed in the manual.
2. Extract cytosolic fraction.
3. Isolate DNA from whole cell extract and cytosolic extract.
4. Load the PCR reaction mix and samples.
5. Set up the PCR Amplification.
6. Export the results.
7. Calculate mtDNA leakage.