Telomere length (TL) measurement is central to many biomedical research, population, and epidemiology studies, with promising potential as a clinical tool. Various assays are used to determine TL, depending on the type and size of the sample. We describe the detailed optimization of a monochromatic multiplex realtime quantitative PCR (MMqPCR) assay for relative TL using the LightCycler 480. MMqPCR was initially developed using a different instrument with many separate reagents. Differences in instrument performance, reagents, and workflow required substantial optimization for the assay to be compatible with the LightCycler 480. We optimized the chemistry of the assay using a purchased one-component reaction mix and herein describe sources of variability and quality control relevant to the MMqPCR TL assay on any instrument. Finally, the assay was validated against other TL assays, such as terminal restriction fragment, Southern blot, and flow fluorescent in situ hybridization. The correlations obtained between data from MMqPCR and these assays (R 2 Z 0.88 and 0.81) were comparable to those seen with the monoplex version (R 2 Z 0.85 and 0.82) when the same samples were assayed. The intrarun and interrun CV ranged from 4.2% to 6.2% and 3.2% to 4.9%, respectively. We describe a protocol for measuring TL on the LightCycler platform that provides a robust high-throughput method applicable to clinical diagnostics or large-scale studies of archived specimens.

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Our results indicate that from birth to 3 years of age, the LTL in HEU children is not negatively affected by exposure to maternal HIV infection and cART, at least not to the regimens used within this Canadian cohort, a reassuring finding.

ABSTRACT Background Combination antiretroviral therapy (cART) during pregnancy prevents vertical transmission, but many antiretrovirals cross the placenta and several can affect mitochondria. Exposure to maternal HIV and/or cART could have long-term effects on children HIV-exposed uninfected (CHEU). Our objective was to compare blood mitochondrial DNA (mtDNA) content in CHEU and children HIV-unexposed uninfected (CHUU), at birth and in early life. Methods Birth and early life (0-3y) whole blood mtDNA content was compared cross-sectionally between CHEU and CHUU children. Longitudinal changes in CHEU mtDNA content was also evaluated. Results At birth, CHEU status and lower gestational age were associated with higher mtDNA content. These remained independently associated with mtDNA content in multivariable analyses, whether considering all infants, or only those born at term. Longitudinally, CHEU mtDNA levels remained unchanged during the first six months of life, and gradually declined thereafter. A separate age and sex-matched cross-sectional analysis (n=214CHEU:214CHUU) illustrates that the difference in mtDNA between the groups remains detectable throughout the first 3 years of life. Conclusion The persistently elevated blood mtDNA content observed among CHEU represents a long-term effect, possibly resulting from in utero stresses related to maternal HIV and/or cART. The clinical impact of altered mtDNA levels is unclear.