Telomere length measurement by qPCR – Summary of critical factors and recommendations for assay design

Abstract

Research in the last decade has explored the length of telomeres, the protective ends of eukaryotic chromosomes, as a biomarker for the cumulative effects of environmental exposures and life experiences as well as a risk factor for major diseases. With a growing interest in telomere biology across biomedical, epidemiological and public health research, it is critical to ensure that the measurement of telomere length is performed with high precision and accuracy. Of the several major methods utilized to determine telomere length, quantitative PCR (qPCR) remains the most cost-effective and suitable method for large-scale epidemiological and population studies. However, inconsistencies in recent reports utilizing the qPCR method highlight the need for a careful methodological analysis of each step of this process. In this review, we summarize each critical step in qPCR telomere length assay, including sample type selection, sample collection, storage, processing issues and assay procedures. We provide guidance and recommendations for each step based on current knowledge. It is clear that a collaborative and rigorous effort is needed to characterize and resolve existing issues related to sample storage, both before and after DNA extraction, as well as the impact of different extraction protocols, reagents and post extraction processing across all tissue types (e.g. blood, saliva, buccal swabs, etc.) to provide the needed data upon which best practices for TL analyses can be agreed upon. Additionally, we suggest that the whole telomere research community be invited to collaborate on the development and implementation of standardized protocols for the assay itself as well as for reporting in scientific journals. The existing evidence provides substantial support for the continuation of telomere research across a range of different exposures and health outcomes. However, as with any technological or methodologic advance in science, reproducibility, reliability and rigor need to be established to ensure the highest quality research.

Reliable blood cancer cells' telomere length evaluation by qPCR

“…Another crucial aspect of telomere length measurement is DNA quality. It is established that one of the primary requests for qPCR‐based techniques in general, and for telomere qPCR in particular, is the use of DNA of high quality . Indeed, we verified that DNA degradation strongly influences telomere length measurements (Figure ).…”

“…The main hurdle in using qPCR‐based techniques to explore cancer cells relies on the selection of an appropriate reference gene . Ploidy abnormalities and chromosome rearrangements are commonly associated with cancer development, making it very likely to select a reference gene that is amplified or lost .…”

“…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 …”

Stress reactivity elicits a tissue-specific reduction in telomere length in ageing zebrafish (Danio rerio)

“…Failure to detect telomere shortening in brain tissues could be a consequence of the lower rate of cell renewal and neurogenesis in the adult brain compared to heart making detection difficult (39,63,69).The lack of consistency in zebrafish findings highlights possible differences in the technique used to assess telomere length, on the rate of telomere attrition between strains (40,41) and animal facilities, which should all be carefully considered. Furthermore, we have combined data from a sibling populations of young and ageing zebrafish, thus allowing for samples to be processed simultaneously, which assures similar storage time and reagents used, critical consideration for the study of telomere biology (71).…”

Universal assay for measuring vertebrate telomeres by real-time quantitative PCR

“…The UCE primer pairs reported here can facilitate robust measurements of telomere lengths in vertebrate study systems, even when limited or no genomic sequence data is available. It is important to establish appropriate tissue collection protocols for each new organism because telomere dynamics vary between tissue types 16 . In addition, the biological relevance of shorter telomere lengths in each organism requires knowledge of other health, age and demographic parameters.…”