Microarray technology is the practical offshoot of the Human Genome Project. The principal goal of the Human Genome Project was to determine the DNA sequence of the entire human genome and, in so doing, identify all human genes. As was recently announced, this historical milestone is nearly complete, giving scientists a nearly complete glossary of human genes to follow as they delve into the secrets of genetic function and malfunction. Genes within the genome only come into play in a cell when they are "expressed." The differences in gene expression among cells is what makes them unique, (i.e. what distinguishes a neuron from a kidney cell), and what allows them to perform their myriad of normal functions. Surprisingly, the human genome consists of only approximately 33,000 genes, a very small number considering the complexity of the human organism.

To measure gene expres-sion of nearly all the genes in the human genome simultaneously, scientists start with an ordinary microscope slide and, using a specialized printing device, deposit tens of thousands of "microspots" onto the slide. Each microspot contains a different gene, and each slide can hold about 30,000 spots. In this way nearly the entire human genome can be printed on a single slide.

A microarray experiment consists of taking normal or diseased tissue, isolating the expressed genes from that tissue, "tagging" the genes with a fluorescent dye, and placing the tagged genes onto the microarray. If a gene is expressed in the tissue it will stick to the microspot on the microarray where that gene is printed and the spot will then "light up" with fluorescent signal. In this way the genes expressed in the tissue can be determined simply by noting which spots light up on the array.

"Using this technology we can answer the question, 'What are all the genes on or turned off in this tissue?', which is to say, 'What is the totality of molecular events that define this state?" Centola said. "Now think what can be done by applying this technology to a tumor sample from a cancer patient, a developing arterial plaque from a heart disease patient, or joint tissue from an arthritis patient. It affords an unprecedented understanding of disease pathogenesis and therefore disease treatment."

Microarray technology is already yielding significant refinement of clinical descriptions of disease states. At present, a given disease is classified based on a collection of clinical symptoms and changes in normal physiology. When clinical diagnostic data and microarray data are combined, subtle differences among patients with a given disease can be observed. The most important point in this regard is that novel treatments can then be developed that target these differences, increasing drug effectiveness and safety.

Take diffuse large B cell lymphoma, for example. Forty percent of patients respond to chemotherapy; however, sixty percent do not respond and succumb to this often fatal disorder.

Using microarrays to refine disease classification researchers studying this disease asked the question, "Can we identify a difference between the responders and non-responders?" A microarray series on 100 patients revealed differences in the genes expressed in patients' tumors, which directly accounted for therapeutic response. There was a clear difference in the genes expressed in the tumors of responders and non-responders. This ability to see which patients will and won't respond to standard therapy can now be used as a diagnostic tool to help physicians provide optimal therapy. Also, by defining the genes that are uniquely expressed in non-responders, pharmaceutical companies can now experiment with agents that target these genes and produce therapies that will kill the tumors in these patients.

Another revolutionary impact microarrays will have on clinical medicine is the ability to aid in refining clinical trials of new drugs. To test the effectiveness of a new drug in a clinical trial, a pharmaceutical company must strictly define the population of patients likely to be helped by the drug before the trial begins. Since subtypes of disease can now be defined, drug effectiveness in these subpopulations of patients can be evaluated.

"This is a wonderfully effective iteration of events: use microarrays to define the molecular basis of drug nonresponsiveness, create custom therapies based on this understanding, and test these new drugs in a way that will allow effectiveness in these subpopulations to be accessed, yielding more powerful, specifically-targeted therapeutics," Centola said.

With data sets this large and complex, Centola and his staff must work hand-in-hand with researchers. "We are more an 'adjunct collaborator' than a core lab," Centola said. "We can determine for a researcher which genes are expressed and which are not, but the researchers we are working with provide the most significant contribution, the scientific intuition that comes from a lifetime of experience in the field of study necessary to determine the meaning of the gene profiles."

An added utility of the OMRF microarray research facility is that it fabricates custom microarrays to suit the given needs of the scientists it serves. Their bar-coded, biorobotic production stream is similar to that of a small pharmaceutical company. "This is about as high-throughput a facility as you can find in an academic setting," Centola said with a smile.

The entire facility occupies little more space than a large walk-in closet. With the equipment currently available at OMRF, Centola's staff can array 5,000 genes per slide (42 slides per run) in about two days. The technology has changed at such a rapid pace that the robot they purchased just eight months ago has already been superceded.

"The near completion of the human genome has made available a much larger number of genes. We now need [a microarrayer] capable of printing the full complement of human genes, approximately 30,000 on one slide, and these have only recently become available," Centola said. "We hope to purchase one soon."

In addition to this work, the microarray research facility has organized a consortium of OMRF scientists including Drs. J. Donald Capra, Linda Thompson, John Harley, Paul Kincade and Kenneth Kaufman, focused on identifying human genes that regulate the immune system.

"Optimally, we want to identify a significant number of the immunologically-relevant genes that have not been previously defined, add those genes onto our arrays, and in this way enhance the relevance in our studies of normal immune system function and dysfunction." said Centola. "OMRF is one of the leading research institutions in the world in the field of immunology, and this speaks directly to that strength and insures our future success."

Centola's colleague in the microarray research facility, Dr. Patrick Cooke, is an expert in bioinfomatics and biorobitic software and hardware. He also has the business background to establish successful collaborative projects with scientists at OMRF and other institutions.

"Although we have only been working together for eight months, it seems like we have known each other for years," Centola said of Cooke. "We have an outstanding working relationship, and it has paid off in success after success on our projects."

Cooke received his Ph.D. degree in microbiology from the University of North Texas in Denton, where he worked as a teaching assistant and laboratory coordinator before coming to OMRF in May.

The facility is also staffed by Dr. Bart Frank, an immunologist and expert in the field of autoimmunity; Dr. Craig Cadwell, a molecular oncologist who recently came to the OMRF from St. Jude's Research Hospital in Memphis; Jeanne Osban, a molecular biologist; and two new members of the staff, Midge Cary and Eric Brown, who came to the facility to learn about microarrys before beginning graduate school in the fall.

"In a short time we have put together a very powerful team," Centola said. "Dr. Cooke is principally responsible for building the microarray production facility and performing data analysis. Midge and Eric, who joined us less than two months ago, have already made significant breakthroughs in their studies of immune cell isolation that have been key to our studies of inflammatory arthritis. Drs. Frank and Cadwell, as well as Jeanne Osban, provide the expertise necessary to deal with the complexity of projects that are ongoing in the facility."

The power of this technology is well understood by biomedical funding agencies. Centola and his colleagues have already received funding through OMRF's IDeA grant with the National Institutes of Health, the National Association of Rare Disorders, and two corporate collaborations. In addition they have contributed in nine separate scientists' recent grant submissions.

Centola received his B.Sc., M.A. and Ph.D. degrees from the University of California, Santa Barbara. He came to OMRF in 2000 as an assistant member in the Arthritis and Immunology Research Program. Centola's previous position was that of senior staff fellow in the National Institutes of Health's Arthritis and Rheumatism Branch.

What made Centola leave the NIH and come all the way to Oklahoma and OMRF?

"Definitely, it was the collegiality of the scientists," Centola said thoughtfully. "I had never seen the top tier of scientists like this looking out for everyone's needs. They really want your science to work. Selflessness, kindness and friendliness. I saw all of those things. I knew I would have a much better chance of success in this environment and could create collaborations that would yield results."

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