Quantifying Mitochondrial DNA Abundance

Total DNA was isolated from cells using Blood and Cell culture kit (QIAGEN), and then subject to qPCR using QuantiTect SYBR Green (QIAGEN). Quantitative PCR was performed using nuclear primers (GAPDH or Tert) and mtDNA primers (mt-CO2, mt-ND2, mt-CO1 and D-loop). Three technical replicates were performed for each biological sample, and the mtDNA abundance of each replicate were normalized against nuclear DNA using the 2^−ΔΔCT method.
For isolation of DNA from mouse plasma, the QIAamp DNA Blood Mini Kit (QIAGEN) was used. Briefly, 100–200 µl mouse plasma was subjected to total DNA extraction. Then the eluted DNA was diluted (1:5) to perform the qPCR experiment.
The related primers used are listed as follows: m-nucDNA GAPDH-DNA-s: GGACCTCATGGCCTACATGG; m-nucDNA GAPDH-DNA-as: TAGGGCCTCTCTTGCTCAG. m-mtCO2-s: TAGGGCACCAATGATACTGAAG; m-mtCO2-as: CTTCTAGCAGTCGTAGTTCACC. m-mtND2-s: AACCCACGATCAACTGAAGC; m-mtND2-as: TTGAGGCTGTTGCTTGTGTG. m-mtCO1-s: CTGAGCGGGAATAGTGGGTA; m-mtCO1-as: TGGGGCTCCGATTATTAGTG. m-mtDNA D loop1-s: AATCTACCATCCTCCGTGAAACC; m-mtDNA D loop1-as^{40 (link)}: TCAGTTTAGCTACCCCCAAGTTTAA. m.mtDNA D loop2-s: CCCTTCCCCATTTGGTCT; m-mtDNA D loop2-as: TGGTTTCACGGAGGATGG. m-mtDNA D loop3-s: TCCTCCGTGAAACCAACAA; m-mtDNA D loop3-as: AGCGAGAAGAGGGGCATT. m-nucDNA Tert-s: CTAGCTCATGTGTCAAGACCCTCTT; m-nucDNA Tert-as: GCCAGCACGTTTCTCTCGTT.

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Zhao Y., Sun X., Hu D., Prosdocimo D.A., Hoppel C., Jain M.K., Ramachandran R, & Qi X. (2019). ATAD3A oligomerization causes neurodegeneration by coupling mitochondrial fragmentation and bioenergetics defects. Nature Communications, 10, 1371.

Publication 2019

Blood and Cell culture kit QuantiTect SYBR Green QIAamp DNA Blood Mini Kit

Biological Blood Cell culture Cells Gapdh Isolation Mouse Mtdna Plasma Primers Replicate Sybr green Tert

Corresponding Organization :

Other organizations : Case Western Reserve University, University School

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Variable analysis

independent variables

Isolation of total DNA from cells using Blood and Cell culture kit (QIAGEN)
Dilution of eluted DNA from mouse plasma (1:5)

dependent variables

Quantitative PCR (qPCR) measurements of mtDNA abundance (mt-CO2, mt-ND2, mt-CO1, D-loop) normalized against nuclear DNA (GAPDH or Tert)

control variables

Three technical replicates for each biological sample
Use of nuclear primers (GAPDH or Tert) as reference for normalization

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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