Anatomy of a Comparative Gene Expression Study
Disclaimer: I'm a computer scientist, not a medical doctor. If you're interested in taking advantage of experimental diagnostic and therapeutic technologies, ask your doctor or visit, e.g., NCI's CancerNet web site.
DNA microarrays are perfectly suited for comparing gene expression in different populations of cells. The hows and whys of such an experiment provide insight into the power of microarrays, their limitations, and the kinds of biological questions which they can help to answer.
The illustration below shows the steps that make up a comparative cDNA hybridization experiment. Click on any of the steps in the image to jump to an explanation of how and why it is performed. Biotechnology terms which are not explained on this page are linked to a glossary, so only a minimal knowledge of modern biology is required. Enjoy!
If your browser can display Flash animations, Professor A. Malcolm Campbell of Davidson College has produced an animated description of comparative hybridization. Requests to use the animation should be directed to Dr. Campbell at macampbell@davidson.edu.
The major steps of a comparative cDNA hybridization experiment are
1. Choosing Cell Populations
The goal of comparative cDNA hybridization is to compare gene transcription in two or more different kinds of cells. We will describe some experiments of particular interest; however, the possibilities for informative comparative transcription studies are limited only by the investigator's imagination.
Tissue-specific Genes
Cells from two different tissues (say, cardiac muscle and prostate epithelium) are specialized for performing different functions in an organism. Although we can recognize cells from different tissues by their phenotypes, it is not known just what makes one cell function as smooth muscle, another as a neuron, and still another as prostate. Ultimately, a cell's role is determined by the proteins it produces, which in turn depend on its expressed genes. Comparative hybridization experiments can reveal genes which are preferentially expressed in specific tissues. Some of these genes implement the behaviors that distinguish the cell's tissue type, while other controlling genes make sure that the cell only performs the functions for its type.
Regulatory Gene Defects in Cancer
Genetic disease is often caused by genes which are inappropriately transcribed -- either too much or too little -- or which are missing altogether. Such defects are especially common in cancers, which can occur when regulatory genes are deleted, inactivated, or become constitutively active. Unlike some genetic diseases (e.g. cystic fibrosis) in which a single defective gene is always responsible, cancers which appear clinically similar can be genetically heterogeneous. For example, prostate cancer (prostatic adenocarcinoma) may be caused by several different, independent regulatory gene defects even in a single patient. In a group of prostate cancer patients, every one may have a different set of missing or damaged genes, with differing implications for prognosis and treatment of the disease.
Comparative hybridization can serve two purposes in studying cancer: it can pinpoint the transcription differences responsible for the change from normal to cancerous cells, and it can distinguish different patterns of abnormal transcription in heterogeneous cancers. Understanding the diverse basis of a cancer is crucial for inventing therapies targeted to the different varieties of the disease, so that each patient receives the most appropriate and effective treatment.
Cancers are common examples of genetically heterogeneous diseases, but they are by no means the only ones. Diabetes, heart disease, and multiple sclerosis are among the diseases for which genetic risk factors are known to be heterogeneous.
Cellular Responses to the Environment
How does a cell adapt to changes in its environment? Cells survive in the face of changes in temperature and pH, changing nutrient availability, and the presence of environmental toxins and ionizing radiation. Usually, a change in environment requires that expression of some genes be turned up or down so that the organism can respond appropriately. For example, common yeast has been extensively studied to understand how it switches between metabolizing sugars into ethanol and ethanol, in turn, into acetic acid (this is why wine with active yeast eventually becomes vinegar). The move from one metabolic state to the other, called diauxic shift, involves shutting down genes for processing sugars and activating others for processing ethanol, as well as a general stress response due to the greater difficulty of deriving energy from ethanol.
Comparative hybridization experiments can point out genes whose transcription changes in response to an environmental stimulus. In the simplest experiment, a population of cells is subjected to the stimulus and allowed to reach a steady state of transcription. Transcription levels in the altered cells can then be compared to those in a control population. A more informative experiment subjects cells to a change, then takes samples of the cell population at successive points in time. In this way, the experimenter can watch as the gene transcription patterns change from the old to the new steady state. Temporal studies can identify not only genes whose transcription changes but also the order of the changes, providing evidence about which genes control the response directly and which are only indirectly affected by it.
Environmental changes of interest also include the introduction of signaling molecules, such as hormones, interleukins, and interferons, as well as the actions of drugs. All these molecules stimulate a change in a cell's behavior (including possibly its death). While some of the changes may be mediated purely at the protein level, others require new transcription which can be detected by comparative hybridization.
Cell Cycle Variations
Even in a stable environment, cells undergo DNA replication, mitosis, and eventually death. These activities require quite different gene products, such as DNA polymerases for genome replication or microtubule spindle proteins for mitosis. A cell's genes encode the "programs" for these activities, and gene transcription is required to execute those programs. Comparative hybridization can be used to distingish genes that are expressed at different times in the cell cycle. In this way, the pathways responsible for controlling basic life processes can be uncovered.
2. mRNA Extraction and Reverse Transcription
Genes which code for protein are transcribed into messenger RNA's (mRNA's) in the cell nucleus. The mRNA's in turn are translated into proteins by ribosomes in the cytoplasm. The transcription level of a gene is taken to be the amount of its corresponding mRNA present in the cell. Comparative hybridization experiments compare the amounts of many different mRNA's in two cell populations.
To prepare mRNA for use in a microarray assay, it must be purified from total cellular contents. mRNA accounts for only about 3% of all RNA in a cell [1], so isolating it in sufficient quantity for an experiment (1-2 micrograms) can be a challenge. Common mRNA isolation methods take advantage of the fact that most mRNA's have a poly-adenine (poly(A)) tail. These poly(A) mRNA's can be purified by capturing them using complementary oligodeoxythymidine (oligo(dT)) molecules bound to a solid support, such as a chromatographic column or a collection of magnetic beads.
Captured mRNA's are still difficult to work with because they are prone to being destroyed. The environment is full of RNA-digesting enzymes (there are some on your fingers, your keyboard, your mouse, and every other exposed surface around you right now), so free RNA is quickly degraded. To prevent the experimental samples from being lost, they are reverse-transcribed back into more stable DNA form. The products of this reaction are called
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