Microarrays demystified - NCT Update

Environmental Health Perspectives, March 15, 2004 by Jennifer Medlin

Scientists are continually searching for new, better, and faster ways to determine which chemicals found in the environment cause adverse health effects, and how they do so. At the same time, the development of new drug therapies depends upon a clearer understanding of how environmental agents cause genetic changes that lead to disease. Since 1997, the NIEHS Microarray Group has been using microarrays to analyze changing patterns of gene expression across the entire genome, studying thousands of affected genes at a time and revolutionizing the way that toxicologic problems are investigated. Today, microarray "chips" allow researchers to complete within a day or so an experiment that would once have taken months using traditional assays focusing on just one gene at a time.

One goal of toxicogenomics is to use gene expression as a highly sensitive and informative marker for toxicity. Using microarrays, researchers can quickly and accurately screen for large numbers of gene expression responses to toxic substances (alone or in mixtures), determine if toxic effects occur at low-dose exposures, highlight vulnerable tissue or cell types, and begin to extrapolate effects from one species to another. Over time, researchers hope to identify genes associated with the development of environmentally caused diseases, including immune dysfunction and cancer, as well as pulmonary, liver, and neurologic diseases.

The Equipment

To make the type of microarray chips used by the NIEHS group, up to 20,000 complementary DNAs (cDNAs) or oligonucleotides are spotted onto a small glass substrate using high-speed robotics and mechanical contact-printing pens. To test a particular toxicant, RNA from treated and control samples is reverse-transcribed into cDNAs while incorporating fluorescent tags. If a red fluorescent tag is used for the treated sample, then a green fluorescent tag will be used for the control sample (or vice versa).

The two fluorescent-labeled cDNA groups are combined onto a microarray chip that contains spots of gene fragments complementary to the labeled cDNAs. An overnight hybridization step allows the labeled cDNAs to find and bind to their complementary gene fragments. Laser scanners detect the red and/or green fluorescent signals of the spotted gene fragments to reveal the relative abundance of the treated and control cDNAs, and hence the relative abundance of the original RNA transcripts. The resulting patterns of color form a gene expression profile, or signature, that points out a possible toxic condition.

According to center director Richard Paules, the first human "ToxChip" microarrays created in the lab contained only the genes believed to be critically involved in toxicity--about 2,000 in all. By the year 2000, researchers were using larger chips with 7,000 clones of rat DNA. "Our goal was to continue to increase our coverage to represent [a] whole genome on a chip," Paules explains. Now chips containing 20,000 elements are available for human, mouse, and rat DNA. Paules says the group has processed more than 12,000 chips since its inception.

The group's laboratory sports a phalanx of sophisticated, often custom-made equipment for fabricating microarrays, scanning the images they produce, and analyzing and archiving the resulting data. A high-speed robotic arrayer prints 96 chips at once, dipping a printer head with 32 pins into a 384-well plate, with each well containing the genetic material that is deposited onto the slide. "We adjusted humidity and temperature controls and introduced liquid-handling robotics to make the spots consistent and uniform," explains microarray lab manager Jeff Tucker, whose background is in biomedical engineering.

Though the group first used its own custom-made chips exclusively, it now for the most part uses chips made by Agilent Technologies, based in Palo Alto, California. Agilent's 20,000-clone human chips are precisely uniform with perfectly circular spots, Tucker says, giving more gene coverage and higher-quality data overall.

As the chips have become more advanced, so has the technology necessary to interpret the data they produce. Three laser scanners collect images produced by the fluorescent dyes used to label RNA on the microarrays. The newest scanner, produced by Agilent Technologies, can scan 48 slides in one carousel run with a time of 7 minutes per slide. The Agilent scanner complements another scanner, manufactured by Axon Instruments of Union City, California, which scans one slide in 15 minutes. (By comparison, the lab's original laser scanner took two hours to scan one slide.) Once scanned, the images are statistically analyzed and combined with clone identification information using commercial software.

The Experts

Analyzing massive amounts of data and converting them to useful information requires professionals skilled in bioinformatics, a multidisciplinary field that combines biology, genetics, information science and technology, statistics, and mathematics. "We use informatics to manage high-output biological data, then analyze and interpret it." explains bioinformatics manager Pierre Bushel, a molecular biologist and informatics expert.