Method comparison studies of telomere length measurement using qPCR approaches: A critical appraisal of the literature

Abstract

Use of telomere length (TL) as a biomarker for various environmental exposures and diseases has increased in recent years. Various methods have been developed to measure telomere length. Polymerase chain reaction (PCR)-based methods remain wide-spread for population-based studies due to the high-throughput capability. While several studies have evaluated the repeatability and reproducibility of different TL measurement methods, the results have been variable. We conducted a literature review of TL measurement cross-method comparison studies that included a PCR-based method published between January 1, 2002 and May 25, 2020. A total of 25 articles were found that matched the inclusion criteria. Papers were reviewed for quality of methodologic reporting of sample and DNA quality, PCR assay characteristics, sample blinding, and analytic approaches to determine final TL. Overall, methodologic reporting was low as assessed by two different reporting guidelines for qPCR-based TL measurement. There was a wide range in the reported correlation between methods (as assessed by Pearson’s r) and few studies utilized the recommended intra-class correlation coefficient (ICC) for assessment of assay repeatability and methodologic comparisons. The sample size for nearly all studies was less than 100, raising concerns about statistical power. Overall, this review found that the current literature on the relation between TL measurement methods is lacking in validity and scientific rigor. In light of these findings, we present reporting guidelines for PCR-based TL measurement methods and results of analyses of the effect of assay repeatability (ICC) on statistical power of cross-sectional and longitudinal studies. Additional cross-laboratory studies with rigorous methodologic and statistical reporting, adequate sample size, and blinding are essential to accurately determine assay repeatability and replicability as well as the relation between TL measurement methods.

Stress and telomere shortening: Insights from cellular mechanisms

“…Because qPCR can be performed with high throughput platforms with relatively small amount of specimen at low cost, it remains the most widely used method for large-scale epidemiological and population studies. However, the high sensitivity of this assay format to the impacts of preanalytical and analytical factors cast some doubts on the accuracy and reproducibility of this method ( Boardman et al, 2014 ; Cunningham et al, 2013 ; Dagnall et al, 2017 ; Denham et al, 2014 ; Hofmann et al, 2014 ; Lin et al, 2018 ; Raschenberger et al, 2016 ; Lindrose et al, 2021 ). Sample sources, DNA extraction methods and storage conditions, PCR conditions, assay re-agents and data analysis methods have all been reported to influence the final data output.…”

Exposure to food insecurity increases energy storage and reduces somatic maintenance in European starlings ( Sturnus vulgaris )

“…The ICC across individuals was 0.77, including treatment week as a fixed factor. Since true TL was presumably changing over the course of the study, 0.77 represents a minimum estimate of the technical repeatability of the TL measurements and suggests measurement precision was acceptably high [42]. All telomere measurements were conducted blind to treatment.…”

Impact of Amplification Efficiency Approaches on Telomere Length Measurement via Quantitative-Polymerase Chain Reaction

“…Most epidemiological studies utilizing TL measurements generated using qPCR either rely on instrument software to estimate amplification efficiency based on a standard-curve, or do not report how efficiency estimates are derived. According to one study, PCR efficiency is the least commonly reported metric in comparative studies utilizing TL measurements generated using qPCR (Lindrose et al, 2021). These norms of practice run counter to new guidelines issued by the Telomere Research Network for studies using qPCR (doi: 10.31219/osf.io/9pzst), as well as the MIQE guidelines for real-time qPCR experiments more broadly (Bustin et al, 2009).…”

“…Concerns expressed about accurate measurement of amplification efficiency for qPCR more broadly (Bustin et al, 2009), have yet to receive substantial attention in telomere research. In fact, details related to PCR efficiency are the least commonly reported metric in comparative studies utilizing TL measurements generated using qPCR (Lindrose et al, 2021). This oversight is concerning, given the increased susceptibility of TL measurements generated via qPCR to variability in amplification efficiency.…”