mtDNA DAMPs were assessed in cell free plasma collected from participants with RHTN and normotensive volunteers. Briefly, cell-free DNA was extracted from 100 μL of plasma using a MagMax Cell-Free DNA Isolation Kit (Applied Biosystems) following the manufacturer’s instructions with minor adaptations (2.5 μL of the MagMax Cell-Free DNA Magnetic Beads per sample was used instead of 5 μL per sample). Cell-free DNA was eluted in 20 μL volumes and aliquots were stored at −80 °C. mtDNA DAMPs were assessed via amplification of DNA within the NADH dehydrogenase subunit 1 and NADH dehydrogenase subunit 6 regions of the mtDNA by real-time polymerase chain reaction as previously described22 (link),27 (link) with minor modifications using a StepOne Plus Real-Time polymerase chain reaction system (ThermoFisher Scientific). DAMP copies were quantified relative to standard samples of known copies (10–50 000 copies). Data are expressed as mtDNA DAMPs per microliter of plasma.
Magmax cell free dna magnetic beads
Manufactured by Thermo Fisher Scientific
Sourced in United States
The MagMax Cell-Free DNA Magnetic Beads is a product designed for the purification of cell-free DNA (cfDNA) from various sample types. The magnetic beads provide a reliable and efficient method for the extraction and concentration of cfDNA, which is commonly used in biomedical research and clinical applications.
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2 protocols using magmax cell free dna magnetic beads
1
Quantifying Mitochondrial DNA Damage Markers
2
Assessing Mitochondrial DNA Damage Markers
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The following methodology was previously described by Butts and associates.64 (link)
mDAMPs were in assessed in serum from human subjects. Briefly, cell-free mitochondrial DNA (cfmtDNA) was extracted from 100 µl of serum using a MagMax™ Cell-Free DNA Isolation Kit (Applied Biosystems, USA) following the manufacturer’s instructions with minor adaptations (2.5 µL of the MagMax™ Cell-Free DNA Magnetic Beads per sample was used instead of 5 µL per sample). CfDNA was eluted in 20 µl volumes and aliquots were stored at −80°C. mDAMPs were assessed via amplification of sequences within the NADH dehydrogenase subunit 1 (ND1) and NADH dehydrogenase subunit 6 (ND6) regions of the mtDNA by Real-Time polymerase chain reaction (PCR) as previously described65 (link),66 (link) with minor modifications using a StepOne Plus Real-Time PCR system (Thermo Fisher Scientific, USA). The mDAMPs assay provided two variables to represent markers of damage: ND1 and ND6.
mDAMPs were in assessed in serum from human subjects. Briefly, cell-free mitochondrial DNA (cfmtDNA) was extracted from 100 µl of serum using a MagMax™ Cell-Free DNA Isolation Kit (Applied Biosystems, USA) following the manufacturer’s instructions with minor adaptations (2.5 µL of the MagMax™ Cell-Free DNA Magnetic Beads per sample was used instead of 5 µL per sample). CfDNA was eluted in 20 µl volumes and aliquots were stored at −80°C. mDAMPs were assessed via amplification of sequences within the NADH dehydrogenase subunit 1 (ND1) and NADH dehydrogenase subunit 6 (ND6) regions of the mtDNA by Real-Time polymerase chain reaction (PCR) as previously described65 (link),66 (link) with minor modifications using a StepOne Plus Real-Time PCR system (Thermo Fisher Scientific, USA). The mDAMPs assay provided two variables to represent markers of damage: ND1 and ND6.
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