Increasing the accuracy and precision of relative telomere length estimates by RT qPCR

Abstract

As attrition of telomeres, DNA caps that protect chromosome integrity, is accelerated by various forms of stress, telomere length (TL) has been proposed as an indicator of lifetime accumulated stress. In ecological studies, it has been used to provide insights into ageing, life history trade-offs, the costs of reproduction and disease. qPCR is a high-throughput and cost-effective tool to measure relative TL (rTL) that can be applied to newly collected and archived ecological samples. However, qPCR is susceptible to error both from the method itself and pre-analytical steps. Here, repeatability was assessed overall and separately across multiple levels (intra-assay, inter-assay and inter-extraction) to elucidate the causes of measurement error, as a step towards improving precision. We also tested how accuracy, defined as the correlation between the "gold standard" for TL estimation (telomere restriction fragment length analysis with in-gel hybridization), could be improved. We find qPCR repeatability (intra- and inter-assay levels) to be at similar levels across three common storage media (ethanol, Longmire's and Queen's). However, inter-extraction repeatability was 50% lower for samples stored in Queen's lysis buffer, indicating storage medium can influence precision. Precision as well as accuracy could be increased by estimating rTL from multiple qPCR reactions and from multiple extractions. Repetition increased statistical power equivalent to a 25% (single extraction analysed twice) and 17% (two extractions) increase in sample size. Overall, this study identifies novel sources of variability in high-throughput telomere quantification and provides guidance on sampling strategy design and how to increase rTL precision and accuracy.

Early‐life telomere length predicts lifespan and lifetime reproductive success in a wild bird

“…DNA purity and concentration was assessed using a NanoDrop (ND‐1000) whilst DNA integrity was assessed by running all samples on a 1% Agarose gel (100 V for 30 min). To measure TL we used a qPCR method based on Criscuolo et al, () and validated for use in M. coronatus by Eastwood et al, () which provided a relative measure of telomere length compared to a control gene. In brief, qPCR reactions were automated in an EpMotion 5075 on 96‐well plates with a total reaction volume of 25 µl.…”

“…Intra‐assay Cq repeatabilities were 0.91 and 0.97 for telomere and GAPDH respectively. qPCR quality control and the calculation of relative telomere length was as per Eastwood et al, (). Relative telomere length was highly repeatable at the interassay repeatability = 0.85 and interextraction repeatability = 0.88 levels.…”

“…Intra-assay Cq repeatabilities were 0.91 and 0.97 for telomere and GAPDH respectively. qPCR quality control and the calculation of relative telomere length was as per Eastwood et al, (2018).…”

“…Eastwood, Mulder, Verhulst, and Peters (2018), DNA was extracted using a modified QIAamp DNA kit (Qiagen) and the au-tomated QIAcube HT instrument. DNA purity and concentration was assessed using a NanoDrop (ND-1000) whilst DNA integrity was assessed by running all samples on a 1% Agarose gel (100 V for 30 min).…”

Reliable blood cancer cells' telomere length evaluation by qPCR

“…The T/S ratio yields a value that is proportional to average telomere length, allowing the determination of relative telomere length . Nevertheless, to obtain accurate, precise, and reproducible data, several factors should be considered …”