Telomere Length Measurement: Key Methodologies

Telomere Length Measurement: Key Methodologies

July 24, 2018 Cancer and telomeres

It is well established among the scientific community that short telomere, specifically the critically short ones, is the variable which determines the onset of senescence and therefore is directly linked to age-related diseases. The pending question is: What do small changes in average or relative TL really mean biologically?

There have been many published studies in the last decades correlating human diseases with progressive telomere shortening. When telomeres become critically short they enter into senescence and it is this trait, not the average telomere length in a cell population, that provides  a meaningful variable.

In order to study telomere biology, several technologies and methodologies have been developed and each has its own advantages and drawbacks.

Most assays measure average or relative telomere length. The critical parameters to consider are reliability, robustness and reproducibility of each methodology. Therefore, robust and reproducible telomere length measurements will predict more accurately the onset of age-related diseases.

There are benefits between simple and high-throughput telomere length measurement methods versus gaining cause-and-effect insights using more precise methods that can provide the whole telomere length distribution.

Q-PCR: Quantitative Polymerase chain reaction. It is used for research and commercial purposes. It is a relatively easy procedure which does not require a high amount of DNA. It determines exclusively relative average telomere length. Another drawback is that such data is not presented in absolute telomere length values of kilobase pairs (kbp). The coefficient variation on the same sample may be higher than 10%.

STELA: Single Telomere Length analysis: It was designed to measure telomeres on individual and on specific chromosomes, and only a small subset of chromosomes has met the criteria needed to allow for the design of primers that yield successful chromosome-specific/chromosome arm-specific telomeric DNA amplification. STELA cannot be used as a large-scale technology.

TRF: Terminal Restriction Fragment. This technique is used only for research purposes. One of the earliest technologies developed used for telomere length testing. It is considered the “gold standard” for telomere length measurement and determines average telomere length. However, it is not a scalable technology and it is limited to the research laboratories.

Flow Fish: This telomere assay could be used as a clinical diagnostic tool. It is the adaptation of QFISH in combination with flow cytometry. The disadvantage is that it cannot be readily adaptable for measure  a wide range of cell types. The biological source is primarily fresh blood samples. Additionally, the clinical utility of the variables measured is limited has it does not measure the whole distribution of telomere length in each cell, neither the percentage of short telomeres. 

HT Q-FISH (High-throughput quantitative fluorescence in situ hybridation): It allows to measure telomere associated variables including mean and median telomere length in kilobase pares, percentage of short telomeres, any percentile and, therefore, the full histogram of telomere length distribution. The intra-assay coefficient of variation is less than 5%. This is truly relevant as it seems that the shortest telomeres, not the average telomere length, are critical for cell viability and chromosome stability. There is a commercially available test that uses HT Q-FISH -CLIA-88/ISO 15189 certified that has been optimized to meet all the needs to be used as a clinical diagnostic tool and able to be scaled up.

The ideal test to measure telomere length should be able to measure each telomere individually - in specific chromosomes and be performed in a reliable, scalable and reproducible way. Nowadays, the technology that fits most of these criteria is HT Q-FISH.

In summary, it is important to establish the cause and effect of telomere length variations and to be able to correlate critical telomere variables with disease given the increased utility for telomere length assessments as a biomarker in research and clinical studies.

Gaining a better understanding of the benefits and drawbacks of various techniques will allow establishing the science of telomere associations with human age related diseases.  

If you want to know more about telomeres and telomere testing please visit www.lifelength.com


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