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Increasing the accuracy and precision of relative telomere length estimates by RT qPCR
- Source :
- Molecular Ecology Resources, 18(1), 68-78. Wiley
- Publication Year :
- 2018
- Publisher :
- Wiley, 2018.
-
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.
- Subjects :
- 0301 basic medicine
DYNAMICS
Accuracy and precision
EVOLUTIONARY BIOLOGY
EPIDEMIOLOGIC RESEARCH
Biology
QUANTITATIVE PCR
VARIES
Real-Time Polymerase Chain Reaction
DNA EXTRACTION METHOD
ECOLOGY
Statistical power
Specimen Handling
03 medical and health sciences
EARLY-LIFE
STATISTICAL POWER
Lysis buffer
Statistics
Genetics
Animals
ASSAY
Passeriformes
repeatability
molecular aging
Ecology, Evolution, Behavior and Systematics
Observational error
Reproducibility of Results
Repeatability
Gold standard (test)
Telomere
telomeres
030104 developmental biology
Blood
Sample size determination
Biotechnology
Subjects
Details
- Language :
- English
- ISSN :
- 14718278 and 1755098X
- Volume :
- 18
- Issue :
- 1
- Database :
- OpenAIRE
- Journal :
- Molecular Ecology Resources
- Accession number :
- edsair.doi.dedup.....6d162ab9a52a1d6126784356b13b7799