February 24, 2009
Agilent Laboratories awarded the 2008 Barney Oliver Prize for Innovation to Bo Curry and Hui Wang, technical contributors in the Molecular Technology Laboratory’s Molecular Tools Department. They developed a microRNA (miRNA) microarray measurement that set a new standard in the scientific community for sensitivity, dynamic range and reproducibility.
Bill Sullivan, Hui Wang, Bo Curry, Darlene Solomon
Sidebars: Microarrays, MiRNA Profiling
Features of new miRNA measurement
Contributions to Agilent's business
About the Barney Oliver Prize
The annual Barney Oliver Prize for Innovation recognizes outstanding technical contributions at Agilent Laboratories that demonstrate creativity, innovation, technical depth, synergy and business value that lead to a useful technical or scientific result.
“We honor Hui and Bo for their research that creates unique and compelling customer value,” said Darlene Solomon, Ph.D., Agilent chief technology officer and vice president or Agilent Laboratories. “Their contributions in chemistry and probe design on top of Agilent’s leading microarray platform allow researchers to study important biological molecules identified only a few years ago.” "We started off with the microarray business, and now we have miRNA and a successful business with double digit profit," said Bill Sullivan, Agilent president and chief executive officer. "This result is an example that differentiates us in the marketplace."
Hui, Bo and their Agilent colleagues faced formidable challenges and questions as they set out to break new ground in miRNA measurement capability.
Would a miRNA measurement solution give Agilent a competitive advantage?
Little was understood about miRNAs when they were first classified as a family of regulatory molecules in 2000. Even as late as 2003 and 2004, leading cancer researchers were taking a wait and see attitude about the importance of miRNAs. When Labs began looking at miRNA measurement in 2003, it wasn’t clear if customers would value miRNA measurement; however miRNA capability could potentially advance and extend the capabilities of Agilent’s leading microarray platform. After early investigation, Labs decided that a project in miRNA measurement really could shape the future of Agilent.
Agilent is a leading provider of DNA microarrays. A microarray is a slide containing hundreds of thousands of spots of DNA called “probes,” which can be used to examine the DNA or RNA content of a tissue sample. The DNA and RNA content reflect the biological character and activity of the tissue sample. Agilent uses a proprietary SurePrint platform to synthesize as many as 1,000,000 high quality probes on a 1-in.x 3-in. glass slide. Because SurePrint technology is “inkjet”-based, it provides a high degree of flexibility that scientists find very useful. Probes can be designed on the customers’ computer, and Agilent can then print them on the microarray for that customers’ exact research needs.
The oldest and most popular use of DNA microarrays is the study of gene expression –which genes in a cell are “turned on” and “off,” and how this might affect the health of the organism. About half of gene expression analyses performed worldwide is for cancer research. Other important gene expression applications include study of cardiovascular disease, immune and inflammatory diseases, and central nervous system disorders.
What were the technical challenges in miRNA profiling?
Small size: MiRNA molecules are very hard to measure because they’re so tiny, just 17 to 25 nucleotides in length. When RNA is extracted from cell samples, only about 0.01 percent of it by weight is miRNA. Their small size also makes them very difficult to label with conventional microarray techniques. Many researchers believed that accurate miRNA measurement required prior size separation from larger RNAs, a process that can be time consuming and can compromise the RNA sample. They also thought that amplification of the miRNAs was required for detection because miRNA is a small fraction of the total RNA and samples are typically available only in very limited quantities.
Similar sequences: Many miRNAs have very similar (homologous) sequences, a property which, along with their small size, makes them very challenging to measure with microarrays.
Expressed with large dynamic range: MiRNAs are expressed with a large dynamic range that is more than four orders of magnitude. So for a given sample, one miRNA sequence can be present in more than 10,000 times greater abundance than that of another miRNA sequence.
Growing database: The data base of miRNAs is growing rapidly. In 2004 there were only about 200 human and human virus miRNAs identified. Now that number is almost 900, and more can still be discovered.
In April of 2007, Agilent introduced the first comprehensive microarray system for profiling human miRNAs, and a year later introduced rat and mouse miRNA microarrays. miRNAs, a class of small non-coding RNAs that are only 19-30 nucleotides long, are estimated to regulate approximately 30 percent of all human genes. Studies have shown that distinct miRNA expression patterns are associated with a number of tumor types and involved in regulating processes such as cell development, metabolism and heart disease. Because miRNAs are key regulators of gene expression, scientists are increasingly interested in measuring them for deeper understanding of biology and to explore their diagnostic, prognostic, and therapeutic potentials. While previously available miRNA microarrays employed microgram quantities of RNA, Agilent’s proprietary direct labeling chemistry produces excellent results from just 100 nanograms of precious RNA sample.
How did Hui and Bo address these challenges?
The success of this platform was the result of complementary skills, shared vision, and team work. Having never used or worked with microarray platforms, Hui disregarded existing beliefs and conventional approaches to RNA labeling, probe design, microarray workflow, and historical expectations about what could be achieved on a microarray platform.
Hui defined the following goals for the new platform: simple, sensitive, quantitative, highly specific, a fit for the customer’s workflow, capable of simultaneous measurement of all known miRNAs and capable of profiling new miRNAs as they continue to be discovered. The ideal measurement also would leverage the strengths of the Agilent microarray platform: high sensitivity and dynamic range, reproducibility, design flexibility and fidelity of probe sequences. Bo shared this vision and joined the project with his in-depth expertise of microarray measurements and data analysis.
“What attracted me to this research was the promise of bringing actual measurement to biology,” said Bo. “The prevailing thinking in the field was, and arguably still is, that you could watch things go up and down, and you could use statistics to decide whether they really did go up or down, but you could never really know how much up or down they went. But now we can build models of the physics of the measurement and reproducibly measure the effects of changes.”
What are the features of this new miRNA measurement?
A very simple protocol to minimize the experimental variation that can occur with any change in sample handling: Hui developed just such a labeling protocol that is easy to do, extremely efficient and extremely reproducible. Most of the tested sequences labeled with 90 to 99 percent efficiency. More importantly, whatever the efficiency, it’s the same over and over again. Their profiling method provides higher performance and higher accuracy with 10-fold less sample and a far simpler protocol than what was available p reviously.
A very direct measurement to minimize any alteration that could change the profile: The labeling uses total RNA without any sample treatment or manipulation. It adds one fluorescence molecule to the end of each miRNA molecule so the level of fluorescence is directly correlated with the number of molecules.
Highly reproducible results: For the microarray probes to measure all RNAs in the complex biological RNA mixture, the interactions between each DNA probe and RNA target have to have similar stability, as indicated by the melting point of the DNA-RNA complex. Together, Bo and Hui developed a method to measure the melting point empirically to select the best set of probes to ensure high specificity and sensitivity in miRNA detection. Bo’s rich experience in and in-depth understanding of microarray measurements enabled a unique microarray design and data analysis method that results in robustly reproducible data even in the event of minor laboratory mishaps.
The arrays owe part of their high reproducibility to Agilent’s strong, custom probe design capability, which allows users to design long probe lengths of 60 nucleotides for a variety of DNA and RNA profiling needs. The probe design for miRNAs contains a hairpin structure that directly abuts the hybridized target miRNA. This hairpin helps stabilize the hybridized target miRNA, and it discourages the binding of RNAs that have very similar sequence to the intended target miRNA, but are too long.
“At the end of this work we have a system that is capable of generating probes that can distinguish miRNAs that differ by only one base or one nucleotide out of the approximately 22 bases,” said Hui. “Usually a single nucleotide mismatch is a deal breaker for this interaction.”
Would the public respond positively to initial results?
At the first public introduction of the miRNA research, Hui’s poster drew a huge audience.
“People couldn’t believe what they were seeing,” said Hui. “They asked me over and over again to make sure there was no typo because for the first time we were demonstrating direct measurement with such low sample input and simple protocol.”
At product introduction in April 2007, the Agilent miRNA assay established itself as the standard to beat, and this differentiation continues to provide significant competitive advantages to Agilent. Research customers (add footnote for info below) are endorsing Agilent’s miRNA capability as critical to furthering their work.
How quickly did the Labs research reach customers?
“I’ve been involved in many projects at HP/Agilent for more than 25 years, and this was one of the fastest significant transfers I’ve ever seen,” said Bo. “Hui started laboratory work in early 2004, and by the summer of 2005, we already had a competitive assay.”
The LSCA genomics business began product development after determining that the Labs miRNA technology was superior to that of an external vendor. Agilent introduced the first comprehensive microarray system for expression profiling of human miRNAs in April 2007, just three years after the first exploratory efforts at Labs.
Would the research contribute to Agilent’s business?
“The development of the miRNA assay has been a key contribution to the success of Agilent’s genomics business and lays a strong foundation for our ability to lead that market for years to come,” said Yvonne Linney, Agilent vice president and general manager of Genomics in LSCA’s Life Sciences Solutions Unit. “With the help of Hui and Bo and the team from Agilent Laboratories and the technology they developed, we have been able to establish ourselves as the No.1 array assay provider for miRNA analysis.
“They developed a great technology that put Agilent in the leadership position for a new emerging market with high margins. They partnered with the business unit to take the technology all the way to successful commercialization, demonstrating a great role model for how new technologies should be developed and commercialized within Agilent.”
“Bo and Hui innovated a better solution, and Yvonne and our Genomics Business did a great job commercializing this technology to change the game for our genomics customers,” said Darlene Solomon, Agilent chief technology officer and vice president, Agilent Laboratories. “At product introduction in April 2007, the Agilent miRNA assay established itself as the standard to beat, and this differentiation continues to command premium pricing for these arrays.”
What are the ongoing challenges?
“We’re still working on this and making it better,” said Bo. The field is moving very rapidly, and customers often want to measure samples that are creative and difficult. Biological samples are very complex, and we’re continually confronted with customer data that we find hard to understand. But we’ve made considerable progress towards convincing the wider community that it can be understood. I think it’s fair to say that our assay and our publications have significantly raised the bar for our competitors.”
What are customers saying?
Washington University: Researchers at the Washington University in St. Louis used Agilent miRNA microarrays to profile uveal melanomas, the second most common form of melanoma. Lori A. Worley, Meghan D. Long, Michael D. Onken and J. William Harbor published an article in the journal Melanoma Research (June 2008, vol. 18, no. 3), finding that miRNA gene expression signatures are good predictors of these melanomas' potential to cause death by metastasizing. (Press release: “Agilent Technologies Delivers Simpler Workflow with miRNA Microarray Labeling and Hybridization Kit,” July 29, 2008)
University of North Carolina School of Medicine: "Tumor-specific predictors based on high-throughput nucleic acid assays offer significant advances over current clinically -derived models," said David Neil Hayes, M.D., assistant professor of medicine, Division of Hematology/Oncology, University of North Carolina School of Medicine. The Hayes team is studying glioblastoma, a highly aggressive type of brain tumor. "My team is on the cusp of being able to correlate a tumor's genetic pattern to clinically-relevant events. We would be hard-pressed to reach this ability in such a short period of time to elucidate a tumor's genetic pattern and understand the regulatory networks between miRNA and gene expression, without the use of Agilent's miRNA and gene expression arrays." (Press release: Agilent Technologies Expands miRNA Microarray Catalog with New Human, Mouse and Rat Assays, April 9, 2008)
Nagoya University: "Lung cancer is the leading cause of cancer-related deaths in Japan," said Dr. Takashi Takahashi, professor of Oncology, Molecular Carcinogenesis at Nagoya University. "We have shown for the first time that let-7 expression is frequently reduced in lung cancers and that alterations in the miRNA expression may have a prognostic impact on the survival of surgically treated lung cancer patients. Agilent miRNA arrays give us the comprehensive miRNA expression profile with excellent performance on sensitivity and accuracy. I expect that the studies of Agilent miRNA array may ultimately provide a foundation for a new paradigm of the involvement of miRNA in human oncogenesis." (Press release: “Agilent Technologies Introduces First Comprehensive Microarray System for Expression Profiling of Human miRNAs,” April 11, 2007)
Genentech: "Agilent's platform for miRNA expression profiling includes a straightforward and easy sample-preparation procedure combined with their well established ink-jet printed oligonucleotide arrays," said Zora Modrusan, scientist and head of the Microarray Laboratory, Molecular Biology at Genentech. "An advantage of their technology compared to the other ones is that a very low amount of starting total RNA sample is required, thus enabling miRNA profiling from clinical samples." (Press release: “Agilent Technologies Introduces First Comprehensive Microarray System for Expression Profiling of Human miRNAs,” April 11, 2007)
About the Agilent Laboratories Barney Oliver Prize for Innovation
The Barney Oliver Prize for Innovation honors Bernard M. Oliver (1916 – 1995), who was a scientist, inventor, and innovator. Agilent Laboratories has awarded the prize annually since 1999 for contributions to Agilent that result from work done in the Labs and that demonstrate Barney's outstanding qualities of creativity, innovation, technical depth, breadth of expertise, and respect for business value. The prize consists of $10,000 after-tax cash and a bronze statue.
Dr. Oliver, known to all as Barney, was a man of enormous intellect, curiosity and vision. When he was 19, he graduated from Stanford University with a B.A. in electrical engineering. A year later he completed an M.S. from the California Institute of Technology, where he earned a Ph.D., graduating magna cum laude at the age of 24.
Barney then joined Bell Telephone Laboratories where he quickly established a reputation for brilliant, creative insights and clever inventions. In 1952, Bill Hewlett and David Packard persuaded Barney, whom they had known since their student days, to join their fledgling operation as director of research. In 1957 he became vice president of Research and Development, and in 1966, he established Hewlett-Packard Laboratories, which he directed until his retirement in 1981.
During his career Barney was awarded 60 patents and authored 71 papers that reflect a remarkable breadth and depth of thought, ideas, and actions spanning physics, mathematics, electronic and electrical engineering, education, and social issues. He was active in the IEEE and served as its president in 1965.
Barney had a lifelong interest in astronomy, and in particular the use of radio telescopes for the search for extraterrestrial intelligence. Between 1982 and 1993 he was the chief engineer of the SETI (Search for Extraterrestrial Intelligence) Institute and member of the SETI board of trustees.
Barney received many awards. In 1986 President Reagan awarded Barney the National Medal of Science for “translating the most profound discoveries of physical and communication science into the electronic, radio, and computer systems which have improved our culture and enriched the lives of all Americans.” In 1997 the SETI Institute established the Bernard M. Oliver Chair, and in 2004 Barney was inducted posthumously into the National Inventors Hall of Fame. Among his academic honors were the Halley Lectureship on Astronomy and Terrestial Magnetism of Oxford University (1984).
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