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Bio-Rad, UCSF Lab Develop Droplet Digital PCR Assay for Telomerase Activity


Bio-Rad is working with Elizabeth Blackburn, a researcher at the University of California, San Francisco School of Medicine and co-founder of Telome Health, to develop a sensitive and high-throughput digital PCR assay to measure telomerase activity in human cells, PCR Insider has learned.

In a poster presentation last week at Cambridge Healthtech Institute's inaugural Integrating Digital PCR conference in Boston, researchers from Bio-Rad's Digital Biology Center in Pleasanton, Calif., provided details of the assay and some early data on its ability to provide an absolute measurement of telomerase activity in cancer cell and immune cell lysates.

According to the presenters, abundant telomerase activity is found in fetal and adult stem cells, germ cells, and cancer, and it is also present at much lower levels in non-pluripotent cells, such as immune cells.

Traditionally, the most accurate and sensitive method for quantifying telomerase activity uses radioactive labeling and PAGE sequencing gels followed by densitometry — a laborious and potentially dangerous procedure.

More recently, researchers have developed the telomerase repeat amplification protocol, or TRAP, assay, which measures the presence of active telomerase on a starting template, which is then amplified by PCR.

For cells showing abundant telomerase activity, this PCR amplification can be performed using a common SYBR Green-based protocol. However, this method falls short in cells with much lower levels of activity.

To address this, Bio-Rad and the Blackburn lab developed a TRAP-based assay that counts single molecules of telomerase-extended templates using the company's QX100 Droplet Digital PCR system.

In Droplet Digital PCR, dilute samples are divided into thousands of separate nanoliter-scale quantitative PCR reaction droplets. Target DNA molecules are distributed among the reaction volumes such that the vast majority contains either one or zero molecules.

The QX100 reader then counts the number of positive and negative reactions, numbers that can be extrapolated to provide an absolute quantification of specific target DNA and normal, background DNA present in the sample.

Using the method, the Bio-Rad and UCSF scientists were able to provide accurate and absolute measurements of telomerase activity in both cancer cell lysates and immune cells lysates. They also found evidence that droplet digital PCR is more sensitive and linear than radiography methods.

Telomerase activity and telomere length in human cells have been linked to a variety of human diseases and conditions, such as cellular aging, diabetes, and cancer.

Thus, the ability to absolutely quantify low levels of telomerase activity may prove useful as a way to detect the onset of these diseases or conditions at a very early stage.

UCSF's Blackburn, who won the 2009 Nobel Prize in Physiology or Medicine for her work on telomere structure and function and discovery of telomerase, and Calvin Harley, a former Geron scientist credited with linking telomeres and telomerase to human cell aging and disease, teamed up with other researchers in 2009 to found Telome Health in an effort to develop assays to measure telomere length for potential diagnostic and health-monitoring applications (PCR Insider, 3/24/2011).

In November, the company said that it expected its first test, a qPCR-based assay to measure average telomere length, to launch in the first quarter of this year. The company has introduced such a test for research purposes, but a clinical version is still in the works, according to the company's website.

It is unclear whether Telome Health is also developing assays to measure telomerase activity as a marker for early disease activity. The company's website makes no mention of telomerase activity assays, and Telome Health representatives did not return a request for comment before deadline. PCR Insider also was unable to reach Blackburn, who is traveling outside the US.

Bio-Rad officials noted that the company's collaboration is with Blackburn's lab at UCSF, and not Telome Health.