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Singapore Team Develops Digital Real-Time PCR Method to Measure Telomeres

NEW YORK – A team of researchers in Singapore has developed a digital real-time PCR-based assay to measure telomere length.

Abnormal telomere length has been linked to a number of aging-related diseases such as diabetes, degenerative disease, and cardiovascular disease, as well as outcomes in various cancers. But the gold standard approach for measuring telomeres requires a large amount of starting DNA and is laborious, making it ill-suited for clinical use.

Researchers led by the National University of Singapore's Lih Feng Cheow devised a new approach dubbed the single telomere absolute-length rapid (STAR) assay to rapidly measure the absolute lengths of telomeres. As they reported Friday in the journal Science Advances, they found the approach to be accurate and sensitive, and reported it could further measure extrachromosomal repeats (ECTRs) found in cancer cells.

"We expect this simple yet comprehensive assay to be widely adopted as a standard measurement for telomeres," Cheow and his colleagues wrote in their paper.

Current approaches for assessing telomere length suffer from drawbacks, according to the researchers. For instance, terminal restriction-fragment (TRF) analysis, the gold standard, relies on Southern blotting, is cumbersome, and not suited for clinical applications. Additionally, real-time qPCR analysis only gives a relative measurement of telomere length, while quantitative fluorescence in situ hybridization can only assess actively dividing cells in metaphase; the peptide nucleic acid hybridization approach PHAST requires specialized equipment; and the STELA approach also relies on Southern blotting and is time-consuming.

The STAR assay the researchers described instead depends on digital PCR to partition the samples so the number of telomere repeats can be measured in nanoliter compartments. They used Fluidigm's 48.770 digital array integrated fluidic circuit and Biomark HD System to develop the assay.

In the assay, PCR amplification leads to an increase in PCR product in proportion to the initial length or copy number of the telomere repeat fragment. The increase in PCR product is reflected in changes in the fluorescence intensity of a DNA binding dye, as captured by real-time monitoring of fluorescence signals.

The assay includes other changes from conventional qPCR, the researchers noted, such as replacing the typical SYBR Green I dye with EvaGreen, as well as changes to the annealing temperature and primer concentrations.

Additionally, to make the assay provide absolute rather than relative lengths, the researchers used different amounts of a synthetic telomere of known length to generate a standard curve against which other telomere lengths can be calculated.

They validated the STAR assay using a panel of human cancer cells that they also analyzed using TRF and other approaches. The average telomere length measured via the STAR assay exhibited high correlation with the gold standard TRF, the researchers reported, indicating the STAR assay is a robust measure of absolute telomere length. They added that the processing time is less than three hours.

The researchers further applied the STAR assay to quantify ECTRs, a feature of cancer cells that use a homologous recombination-based mechanism to preserve their telomeres. The amount of ECTRs could reflect the severity of alternative lengthening of telomeres (ALT) in cancer cells — which is associated with poorer prognosis — and could inform treatment choices.

Using the STAR assay, the researchers profiled four clinical pediatric neuroblastoma samples. In alignment with other studies, they concluded that two samples were ALT-positive and two were ALT-negative, suggesting the STAR assay could be applied to determine telomere maintenance mechanisms within oncology labs.

"With the wide adoption of digital PCR assays in clinical laboratories and the introduction of commercial high-throughput digital PCR platforms that enable real-time reaction monitoring, we envision the STAR assay to be a new paradigm for measuring telomere length as a biomarker," Cheow and his colleagues wrote.

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