eTag, you're it!

Researchers use microarrays to cull from the 30,000 genes identified by the Human Genome Project those that interact with environmental toxicants. Generally, anywhere from 50 to 200 genes stand out and are further scrutinized by time-consuming bioinformatics algorithms. Now a new tool called the eTag Assay System rapidly identifies not only genes but "also related proteins without the need for complex sample preparation and follow-up bioinformatics, eTags further allow scientists to look at multiple concentrations of an environmental toxicant to determine at what dose it becomes toxic, says Sharat Singh, inventor of the eTag chemistry and senior vice president of technology and assay development at Aclara Biosciences in Mountain View, California.

Short for "electrophoretic tags," eTags are small fluorescent molecules linked to nucleic acids or antibodies and are designed to bind one specific messenger RNA or protein, respectively. After the eTag binds its target, a special proprietary enzyme cleaves the bound eTag from the target. The signal generated from the released eTag, called a "reporter," is proportional to the amount of target messenger RNA or protein in the sample.

The eTag reporters are identified by capillary electrophoresis using a capillary-based DNA sequencer. The unique charge-to-mass ratio of each eTag reporter--that is, its electrical charge divided by its molecular weight--makes it show up as a specific peak on the capillary electrophoresis readout.

Unlike microarrays, samples used with the eTag system do not require expensive preparation steps, such as amplification by polymerase chain reaction. Nanogram quantities of cells, tissue, blood, or bodily fluids are mixed directly with eTag reagents in standard 96-well laboratory microplates.

The ability to look at genes and proteins at the same time yields multiple advantages for researchers. Running only one assay means less sample is needed, and time and equipment costs are lower. This ability also increases the specificity of the assay by confirming that certain genes express certain proteins. Finally, because some genes are regulated at the transcriptional level and others at the translational level, measuring genes and proteins gives a better understanding of the regulation of gene expression.

Because of the costs related to running microarrays, researchers often obtain data for just one dose of a given compound. Yet, "it would be enlightening to look at one hundred compounds at ten concentrations and ten time points to fully understand toxic reactions," says Singh. Such a comprehensive experiment would require hundreds of microarrays (at about $300 apiece) and a few months to complete. In contrast, eTags could accomplish the task in days, at a cost of approximately $200.

Fifty different eTags corresponding to 50 reactions can be combined in one well. Aclara offers eTag assay kits to detect certain pathways, such as cytokines and cytochrome P450s associated with toxicity, or they can customize sets of eTags to suit a researcher's needs.

At Stanford University School of Medicine, rheumatologist Paul J. Utz received funding from the National Heart, Lung, and Blood Institute to use eTags to identify cytokines and chemokines as biomarkers for systemic lupus erythematosus and rheumatoid arthritis. In mouse models of rheumatoid arthritis, environmental toxicants such as mercury trigger the production of characteristic autoantibodies, which may also be pathogenic. In past experiments, Utz has relied on homemade protein arrays to detect biomarkers such as autoantibodies and cytokines. However, these protein arrays "are a pain to print, they do not allow us to study all proteins, and the sensitivity is not as good as what we get with eTags," he says. Moreover, Utz adds, eTags "are easier to use and have great flexibility."

Because eTags are small molecules that do not interfere with biological processes, the opportunities for their use are broad, says Singh, ranging from detecting biowarfare agents such as anthrax and smallpox, to discovering biomarkers of cancer. Aclara scientists have created 600 eTags for genes and proteins relevant to toxicology and human illnesses, providing significant coverage of key targets. The ultimate goal is to create 1,000 eTags by the end of 2004, says Singh.

The eTag Assay System

Nanogram quantities of cells, tissue, blood, or body fluids can be mixed directly with eTag reagents in standard 96-well laboratory microplates. Fifty different eTags (small fluorescent molecules linked to nucleic acids or antibodies) corresponding to 50 reactions can be combined in one well.

eTags bind one specific messenger RNA or protein. Then a special proprietary enzyme cleaves the bound eTags from the target. The "reporter" signal generated from the released eTag is proportional to the amount of target messenger RNA or protein in the sample.

A DNA sequencer identifies the eTag reporters by capillary electrophoresis. The unique charge-to-mass ratio of each eTag reporter makes it show up as a specific peak on the capillary electrophoresis readout.