http://www.ncbi.nlm.nih.gov/About/primer/microarrays.html
Microarrays Factsheet National Center for Biotechnology Information About NCBI NCBI at a Glance A Science Primer Databases and Tools Human Genome Resources Model Organisms Guide Outreach and Education News About NCBI Site Map Science Primer: Bioinformatics Genome Mapping Molecular Modeling SNPS ESTS Molecular Genetics Pharmacogenomics Phylogenetics Just the Facts: A Basic Introduction to the Science Underlying NCBI Resources MICROARRAYS: CHIPPING AWAY AT THE MYSTERIES OF SCIENCE AND MEDICINE The proper and harmonious expression of a_large number of genes is a critical component of normal growth and development and the maintenance of proper health. Disruptions or changes in gene expression are responsible_for many diseases. With only a_few exceptions, every cell of the body contains a full set of chromosomes and identical genes. Only a fraction of these genes are turned_on, however, and it is the subset that_is'expressed'that confers unique properties to each cell type.''Gene expression'is_used the term to describe the transcription of the information contained within the DNA--the repository of genetic information--into MESSENGER_RNA (mrna) molecules that are_translated then into the proteins that perform most of the critical functions of cells. Scientists study the kinds and amounts of mrna produced by a cell to learn which genes are_expressed, which in_turn provides insights into how the cell responds to its changing needs. Gene expression is a highly complex and tightly regulated process that allows a cell to respond dynamically both to environmental stimuli and to its own changing needs. This mechanism acts as both an'on/off'switch to control which genes are_expressed in a cell as_well_as a'volume control'that increases or decreases the level of expression of particular genes as necessary. Enabling Technologies Biomedical research evolves and advances not_only through the compilation of knowledge, but_also through the development of new technologies. Using traditional methods to assay gene expression, researchers were able to survey a relatively small number of genes at_a_time. The emergence of new tools enables researchers to address previously intractable problems and to uncover novel potential targets for therapies. Microarrays allow scientists to analyze expression of many genes in a single experiment quickly and efficiently. They represent a_major methodological advance and illustrate how the advent of new technologies provides powerful tools for researchers. Scientists are using microarray technology to try to understand fundamental aspects of growth and development as_well_as to explore the underlying genetic causes of many human diseases. DNA Microarrays: The Technical Foundations Two recent complementary advances, one in knowledge and one in technology, are greatly facilitating the study of gene expression and the discovery of the roles played by specific genes in the development of disease. As_a_result_of the Human Genome Project, there has_been an explosion in the amount of information available about the DNA sequence of the human genome. Consequently, researchers have_identified a_large number of novel genes within these previously unknown sequences. The challenge currently facing scientists is to find a way to organize and catalog this vast amount of information into a usable form. Only after the functions of the new genes are_discovered will the full impact of the Human Genome Project be_realized. The second advance may facilitate the identification and classification of this DNA sequence information and the assignment of functions to these new genes: the emergence of DNA microarray technology. A microarray works by exploiting the ability of a given mrna molecule to bind specifically to, or hybridize to, the DNA template from which it originated. By using an array containing many DNA samples, scientists can determine--in a single experiment--the expression levels of hundreds or thousands of genes within a cell by measuring the amount of mrna bound to each site on the array. With the aid of a computer, the amount of mrna bound to the spots on the microarray is_measured precisely, generating a profile of gene expression in the cell. A microarray is a tool for analyzing gene expression that consists of a small membrane or glass slide containing samples of many genes arranged in a regular pattern. Why are Microarrays Important? Microarrays are a significant advance both because they may contain a very large number of genes and because_of their small size. Microarrays are therefore useful when one wants to survey a_large number of genes quickly or when the sample to be_studied is small. Microarrays may_be_used to assay gene expression within a single sample or to compare gene expression in two different cell types or tissue samples, such_as in healthy and diseased tissue. Because a microarray can be_used to examine the expression of hundreds or thousands of genes at_once it promises to revolutionize the way scientists examine gene expression. This technology is_considered still to be in its infancy; therefore, many initial studies using microarrays have_represented simple surveys of gene expression profiles in a variety of cell types. Nevertheless, these studies represent an important and necessary first_step in our understanding and cataloging of the human genome. As more information accumulates, scientists will_be able to use microarrays to ask increasingly complex questions and perform more intricate experiments. With new advances, researchers will_be able to infer probable functions of new genes based_on similarities in expression patterns with those of known genes. Ultimately, these studies promise to expand the size of existing gene families, reveal new patterns of coordinated gene expression across gene families, and uncover entirely new categories of genes. Furthermore, because the product of any_one gene usually interacts with those of many others, our understanding of how these genes coordinate will become clearer through such analyses, and precise knowledge of these inter-relationships will emerge. The use of microarrays may also speed the identification of genes involved in the development of various diseases by enabling scientists to examine a much larger number of genes. This technology will also aid the examination of the integration of gene expression and function at the cellular level, revealing how multiple gene products work together to produce physical and chemical responses to both static and changing cellular needs . What Exactly is a DNA Microarray? DNA Microarrays are small, solid supports onto which the sequences from thousands of different genes are_immobilized, or attached at fixed locations. The supports themselves are usually glass microscope slides--the size of two side-by-side pinky fingers --but can also be silicon_chips or nylon membranes. The DNA is_printed, spotted, or actually synthesized directly onto the support. The American Heritage Dictionary defines'array'as'to place in an orderly arrangement.''It is important that the gene sequences in a microarray are_attached to their support in an orderly or fixed way, as a researcher uses the location of each spot in the array to identify a particular gene sequence. The spots themselves can be DNA, cdna, or oligonucleotides. An oligonucleotide, or oligo as_it_is commonly called, is a short fragment of a single stranded DNA that_is typically 5 to 50 nucleotides long. Designing a Microarray Experiment: The Basic Steps One might ask, how does a scientist extract information about a disease condition from a dime-sized glass or silicon_chip containing thousands of individual gene sequences? The whole process is based_on hybridization probing, a technique that uses fluorescently labeled nucleic_acid molecules as'mobile probes'to identify complementary molecules--sequences that are able to base-pair with one_another. Each single stranded DNA fragment is_made up of four different nucleotides, adenine (A), thymine (T), guanine (G) and cytosine (C), that are_linked end_to_end. Adenine is the complement of, or will always pair with, thymine, and guanine is the complement of cytosine. So, the complementary sequence to G-T-C-C-T-A will_be C_a-G_g-A_t . When two complementary sequences find each_other--such_as the immobilized target DNA and the mobile probe DNA, cdna, or mrna--they will lock together, or hybridize. Now, consider two cells: cell type 1, a healthy cell, and cell type 2, a diseased cell. Both contain an identical set of four genes, A_b_c, and D. Scientists are_interested in determining the expression profile of these four genes in the two cells types. To do_isolated this, scientists mrna from each cell type and used this mrna as templates to generate cdna with a'fluorescent tag'attached. Different tags (red and green) are_used so_that the samples can be_differentiated in subsequent steps. The two labeled samples are_mixed then and incubated with a microarray containing the immobilized genes A, B_c, and D. The labeled molecules bind to the sites on the array corresponding to the genes expressed in each cell. A DNA Microarray Experiment 1. Prepare your DNA chip using your chosen target DNAS. 3. Incubate your hybridization mixture containing fluorescently labeled cdnas with your DNA chip. 4. Detect bound cdna using laser technology and store data in a computer. 5. Analyze data using computational methods. 2. Generate a hybridization solution containing mixture of fluorescently-labeled cdnas. After this hybridization step is complete, a researcher will place the microarray in a'reader 'or'scanner'that consists of some lasers, a special microscope, and a camera. The fluorescent tags are_excited by the laser, and the microscope and camera work together to create a digital image of the array. This data is_stored then away in a computer and a special program is_used to either calculate the red to green fluorescence ratio or to subtract out background data for each microarray spot by analyzing the digital image of the array. If calculating ratios, the program then creates a table that contains the ratio of the intensity of red to green fluorescence for every spot on the array. For_example, using the scenario outlined above, the computer may conclude that both cell types express gene A at the same level, that cell 1 expresses more of gene B, that cell 2 expresses more of gene C, and that neither cell expresses gene D . But remember, this_is a simple example used to demonstrate key points in experimental design. Some microarray experiments can contain up_to 30,000 target spots. Therefore, the data generated from a single array can mount_up quickly. The Colors of a Microarray Reproduced with permission from the Office of Science Education, the National_institutes_of_health. In_this schematic: GREEN represents Control DNA where either DNA or cdna derived from normal tissue is_hybridized to the target DNA. RED represents Sample DNA where either DNA or cdna is_derived from diseased tissue hybridized to the target DNA. YELLOW represents a combination of Control and Sample DNA where both hybridized equally to the target DNA. BLACK represents areas where neither the Control nor Sample DNA hybridized to the target DNA. Each spot on an array is_associated with a particular gene. Each color_in an array represents either healthy (control) or diseased (sample) tissue. Depending_on the type of array used the location and intensity of a color will tell us whether the gene, or mutation, is present in either the control and/or sample DNA. It will also provide an estimate of the expression level of the gene (s) in the sample and control DNA. Types of Microarrays There_are three basic types of samples that can be_used to construct DNA microarrays--two are genomic and the other is'transcriptomic,'that_is, it measures mrna levels . What makes them different from each_other is_immobilized the kind_of DNA used to generate the array and ultimately, the kind_of information that_is derived from the chip. The target DNA used will also determine the type of control and sample DNA that_is used in the hybridization solution. I. Changes in Gene Expression Levels Determining the level, or volume, at which a certain gene is_expressed is_called microarray expression analysis, and the arrays used in_this kind_of analysis are_called are_called'expression chips.''The immobilized DNA is_derived cdna from the mrna of known genes, and, once_again--at_least in some experiments--the control and sample DNA hybridized to the chip is_derived cdna from the mrna of normal and diseased tissue, respectively. If a gene is overexpressed in a certain disease state, then more sample cdna, as compared to control cdna, will hybridize to the spot representing that expressed gene. In_turn, the spot will fluoresce red with greater intensity than it will fluoresce green. Once researchers have_characterized the expression patterns of various genes involved in many diseases cdna derived from diseased tissue from any individual can be_hybridized to determine whether the expression pattern of the gene from the individual matches the expression pattern of a known disease. If this_is the case, treatment appropriate for that disease can be_initiated. As researchers use expression chips to detect expression patterns --whether or not a particular gene (s_is being_expressed more_or_less under certain circumstances--expression chips may also be_used to examine changes in gene expression over a given period of type, such_as within the cell cycle. The cell cycle is a molecualr network that determines, in the normal cell, if the cell should pass_through its life_cycle. There_are a variety of genes involved in regulating the stages of the cell cycle. Also built into this network are_designed mechanisms to protect the body when this system fails or breaks down due_to mutations within one of the'control genes, 'as is the case with cancerous cell growth. An expression microarray'experiment'could be_designed where cell cycle data is_generated in multiple arrays and referrenced to time'zero.''Analysis of the collected data could further elucidate details of the cell cycle and its'clock,'providing much needed data on the points at which gene_mutation leads to cancerous growth as_well_as sources of therapeutic intervention. In the same way, expression chips can be_used to develop new drugs. For_instance, if a certain gene is overexpressed in a particular form of cancer, researchers can use expression chips to see if a new drug will reduce overexpression and force the cancer into remission. Expression chips could also be_used in disease diagnosis as_well. For_example in the identification of new genes involved in environmentally triggered diseases--such_as those diseases affecting the immune, nervous, and pulmonary/respiratory_systems. II. Genomic Gains And Losses DNA repair genes are_thought to be the body's frontline defense against mutations, and, as_such, play a_major role in cancer. Mutations within these genes often manifest themselves as lost or broken chromosomes. It has_been_hypothesized that certain chromosomal gains and losses are related_to cancer progression and that the patterns of these changes are relevant to clinical prognosis. Using different laboratory methods, researchers can measure gains and losses in the copy number of chromosomal regions in tumor cells. Then, using mathematical models to analyze this data, they can predict which chromosomal regions are most likely to harbor important genes for tumor initiation and disease progression. The results of such an analysis may_be_depicted as a hierarchical treelike branching diagram, referred to as a'tree model of tumor progression.''Researchers use a technique called microarray Comparative Genomic Hybridization, or microarray CGH, to look_for genomic gains and losses, or for a change in the number of copies of a particular gene involved in a disease state. In microarray CGH, large pieces of genomic DNA serve as the target DNA, and each spot of target DNA in the array has_known a chromosomal location. The hybridization mixture will contain fluorescently labeled genomic DNA harvested from both normal (control) and diseased (sample) tissue. So, if the number of copies of a particular target gene has_increased, a_large amount of sample DNA will hybridize to those spots on the microarray that represent the gene involved in_that disease, whereas comparatively small amounts of control DNA will hybridize to those same spots. As a result, those spots containing the disease gene will fluoresce red with greater intensity than they will fluoresce green, indicating that the number of copies of the gene involved in the disease has_gone up. III. Mutations in DNA When researchers use microarrays to detect mutations or polymorphisms in a gene sequence, the target, or immobilized DNA, is usually that of a single gene. In_this case though, the target sequence placed on any given spot within the array will differ from that of other spots in the same microarray, sometimes by only one or a_few specific nucleotides. One type of sequence commonly used in_this type of analysis is_called a Single Nucleotide Polymorphism, or SNP--a small genetic change, or variation, that can occur within a person's DNA sequence. Another difference in mutation microarray analysis, as compared to expression or CGH microarrays, is that this type of experiment only requires genomic DNA derived from a normal sample for use in the hybridization mixture. Once researchers have_established that a SNP pattern is_associated with a particular disease, they can use SNP microarray technology to test an individual for that disease expression pattern in_order_to determine if he or she is susceptible to--at risk of developing--that disease. When genomic DNA from an individual is_hybridized to an array loaded with various SNPS, the sample DNA will hybridize with greater frequency only to specific SNPS associated with_that person. Those spots on the microarray will then fluoresce with greater intensity, demonstrating that the individual being_tested may_have, or is at risk for developing, that disease. In_brief: Microarray Applications Microarray Type Application CGH Tumor Classification, Risk Assessment, and Prognosis Prediction. Expression Analysis Drug Development, Drug Response, and Therapy Development. Mutation/Polymorphism Analysis Drug Development, Therapy Development, and Tracking Disease Progression. NCBI and Microarray Data Management Why is it necessary to have a uniform system that will manage and provide a disbursement point for microarray data? First, consider the amount of data that can potentially be_generated using a single microarray chip. Suppose that chip contains 30,000 spots of target DNA. Researchers interpreting the data generated by that chip would need to know the biological identity of each target --what gene is where; the biological properties of the control and sample DNA; the experimental_conditions and procedures used in setting up the experiment; and finally, the results. While experiments such_as these will undoubtedly push_forward our current understanding of gene expression and regulation, it poses many new challenges in_terms_of data tracking and analysis. What Is GEO? As we have_alluded just to, microarray technology is one of the most recent and important experimental breakthroughs in molecular_biology. Today, proficiency in generating data is fast overcoming the capacity for storing and analyzing it. Much of this information is_scattered across the Internet, or is not even available to the public. As more laboratories acquire this technology, the problem will only get_worse. This avalanche of data requires standardization of storage, sharing, and publishing techniques. In_order_to support the public use and dissemination of gene expression data, the NCBI has_launched the Gene Expression Omnibus, or GEO. GEO represents NCBI's effort to build an expression data repository and online resource for the storage and retrieval of gene expression data from any organisms or artificial source. Many types of gene expression data, such_as those types discussed in_this primer, will_be_accepted and archived as a public data set. COMING SOON... Need help with GEO? How about a quick'how-to'for using various other NCBI data_mining tools? Try GETTING STARTED, a new NCBI resource designed to aid the novice user. GETTING STARTED will show you: Brief descriptions of data that can be found and manipulated using a particular NCBI tool; Shortcuts and tips for getting to where you need to go; Concise explanations of NCBI tool graphics; Insider techniques for conducting database searches; and Simple examples of tool usage Developing MAML: Reading Off the Same Platform Microarray Markup_language, developed by the'MAML'working_group of MGED, the Microarray Gene Expression Database, is a first attempt to provide a standard platform for submitting and analyzing the enormous amounts of microarray expression data generated by different laboratories around the world. The goal of this group, which includes NCBI investigators, is to facilitate the adoption of standards for DNA-array experiment annotation and data representation, as_well_as the introduction of standard experimental controls and data normalization methods. The underlying goal is to facilitate the establishing of gene expression data repositories; comparability of gene expression data from different sources; interoperability of different gene expression databases; and, data analysis software. MAML proposes a framework for describing information about a DNA-array experiment and a data_format for communicating this information, including details about: Experimental design: the set of the hybridization experiments as_a_whole; Array design: each array used and each spot on the array; Samples: samples used, the extract preparation, and labeling; Hybridizations: procedures and parameters; Measurements: images, quantitation, and specifications; and Controls: types, values, and specifications. MAML is independent of the particular experimental platform and provides a framework for describing experiments done on all types of DNA-arrays, including spotted and synthesized arrays, as_well_as oligo and cdna arrays. What's more, MAML provides format to represent microarray data in a flexible way, which allows analysis of data obtained from not_only any existing microarray platforms, but_also many of the possible future variants, including protein arrays . Although the data in GEO is_provided not currently in MAML format, it is NCBI's goal to have_delivered the data in a number of formats, including MAML--soon to be_replaced by a more recent version called MAGEML (Microarray Gene Expression Markup_language. The Benefits of GEO and MAML By storing vast amounts of data on gene expression profiles derived from multiple experiments employing varied criteria and conditions, GEO will aid in the study of functional genomics--the development and application of global experimental approaches to assess gene function. GEO will facilitate the cross-validation of data obtained using different techniques and technologies and will help set benchmarks and standards for further gene expression studies; By making the information stored in GEO publicly available, the fields of bioinformatics and functional genomics will_be_promoted both and advanced; and That such experimental data should be freely accessible to all is consistent with NCBI's legislative mandate and mission: To develop new information_technologies to aid in the understanding of fundamental molecular and genetic processes that control health and disease. The Promise of Microarray Technology in Treating Disease Now_that you understand the concept behind array technology, picture this: a hand-held instrument that a physician could use to quickly diagnose cancer or other diseases during a routine office visit. What if that same instrument could also facilitate a personalized treatment regimen--exactly right for you? Personalized drugs. Molecular diagnostics. Integration of diagnosis and therapeutics. These are the long-term promises of microarray technology. Maybe not today or even tomorrow, but someday. For_the_first_time, arrays offer hope for obtaining global views of biological_processes--simultaneous readouts of all_the body's components--by providing a systematic way to survey DNA and RNA variation. NCBI, by continuing its efforts to provide a standard format for microarray data and to provide free, universal access to_that data, will help the scientific community in making those promises realities. Back to top Revised November 27 2001 NCBI NLM NIH Privacy Statement Disclaimer Accessibility
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