Concurrent Measurement of Mitochondrial DNA Copy Number and ATP Concentration in Single Bovine Oocytes
Abstract
:1. Introduction
2. Materials and Methods
2.1. Collection of Cumulus-Oocyte-Complexes
2.2. TaqMan Primer and Probe Design
2.3. Validation
2.3.1. Oocyte Lysis and Division
2.3.2. ATP Quantification Validation
2.3.3. mtDNA Copy Number Quantification Validation
2.4. Germinal Vesicle and Metaphase II Comparison
2.4.1. Oocyte Lysis and Division
2.4.2. ATP Quantification
2.4.3. mtDNA Standard Preparation
2.4.4. mtDNA Copy Number Quantification
2.5. Statistics and Analyses
3. Results and Discussion
3.1. Standards Quality
3.2. Validation of Procedures for Quantification of ATP and mtDNA Copy Number in Quarter Oocyte Equivalents
3.3. ATP and mtDNA Copy Number in GV versus MII Oocytes
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Name | Sequence | Product Length | Tm |
---|---|---|---|
mtDNA Primer Forward | CCTACAAACGCTCCTTCCAC | 183 | 59 |
mtDNA Primer Reverse | AGAGAATATAGGGCGGTGATTACT | 183 | 59 |
TaqMan Probe | TTGTTGGGGGTAGAGCTAAGTTGGT | --- | 64 |
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Read, C.C.; Bhandari, S.; Moorey, S.E. Concurrent Measurement of Mitochondrial DNA Copy Number and ATP Concentration in Single Bovine Oocytes. Methods Protoc. 2021, 4, 88. https://doi.org/10.3390/mps4040088
Read CC, Bhandari S, Moorey SE. Concurrent Measurement of Mitochondrial DNA Copy Number and ATP Concentration in Single Bovine Oocytes. Methods and Protocols. 2021; 4(4):88. https://doi.org/10.3390/mps4040088
Chicago/Turabian StyleRead, Casey C., Sadikshya Bhandari, and Sarah E. Moorey. 2021. "Concurrent Measurement of Mitochondrial DNA Copy Number and ATP Concentration in Single Bovine Oocytes" Methods and Protocols 4, no. 4: 88. https://doi.org/10.3390/mps4040088