Analysis of Relative Telomere Length and mtDNA Content in Individual Oocytes and Embryos by Quantitative Real-Time...

Oocytes and embryos are highly susceptible to intrinsic and extrinsic stressors, making assays for biomarkers of embryo quality of high importance. This has been technically challenging due to the very small amounts of material in embryos, leading to the use of assays requiring pooled embryos which mask inter-embryo variability. This protocol details a qPCR assay to analyze relative telomere lengths and mtDNA content in individual oocytes and embryos. Each is singly collected and lysed in a minimal volume, then qPCR is used to independently amplify two nuclear (telomere and Rn18S) and one mitochondrial DNA sequence for quantification of relative telomere length or mtDNA content that is normalized for cell number by the reference gene. This methodology enables robust and precise measurements of these molecular biomarkers in physiological contexts where tissue availability is limited. Importantly, this assay allows detection of the natural variability in mtDNA and telomeric DNA content between individual oocytes and embryos.