Impact of DNA Extraction Methods on Quantitative PCR Telomere Length Assay Precision in Human Saliva Samples

Abstract

Telomere length (TL) has emerged as a promising replicative cellular aging marker that reflects both genetic and non-genetic influences. Quantitative PCR (qPCR) TL measurement has been favored as a cost-effective method that can be easily implemented, especially in population studies with limited quantities of source material. However, several recent reports have revealed inconsistencies in telomere length measurements when applying different DNA extraction methods to the same source material. In this study we tested three DNA extraction methods on saliva samples from 48 participants of the National Growth and Health Study (NGHS) collected with DNA Genotek’s Oragene kit. The chosen extraction kits represent three distinct approaches to genomic DNA extraction from lysed cells and we employed two different operators to carry out all assays on the same samples. We measured DNA yield and quality and calculated the between-operator agreement of qPCR TL measurements (intraclass correlation, ICC). Our analyses showed that while both QIAamp and Agencourt DNAdvance had higher agreement between the 2 operators (ICC=0.937, CI [0.891, 0.965] and ICC=0.95, CI [0.911, 0.972] respectively), compared to PrepIT kit (ICC=0.809, CI [0.678, 0.889]), QIAamp extracted DNA samples were notably degraded. Using generalizability theory, we found that the participant-by-extraction-method interaction explained about 10% of total variation in TL, suggesting that TL differences across methods are somewhat participant-specific. Therefore, our results suggest that the among the three DNA extraction methods tested, Agencourt (magnetic bead purification) is the preferred kit, and we also strongly recommend against combining different extraction methods within a study population.