Plate reader

Related Terms

C. elegans, chip, clotting factor VIII, development, disease, DNA, DNA microarray, fluorescence, folate receptor, gene, hemophilia, Huntington's disease, hybridization, hybridize, metastasis, microarray technology, plate reader, probe, prostate cancer, RNA, target.

Background

DNA is located in a compartment of the cell called the nucleus and is packaged in structures called chromosomes. Human cells contain 46 chromosomes, each of which has hundreds of genes. Chromosomes also contain many other regulatory sequences that control how much of a gene will be made, when it will be made, and where in the body it will be made. Genes contain the instructions for making the proteins that do the work in the human body. Variants of a gene are referred to as alleles.
Although all cells in the human body contain the same DNA, cell types in the body vary widely. For example, a skin cell is different from a brain cell in terms of how it looks and what it does. Many of the differences among cells are not caused by differences in the DNA, but rather to differences in the amount and types of genes that the cell expresses. Cells express genes by making RNA, which is then converted into proteins. Each gene produces its own unique RNA. Measuring the amount of different RNAs in a cell can tell researchers how much of each gene is produced by that cell. The sum total of the active genes in a cell, which can be determined by identifying all of the mRNA present within a cell, is called a transcriptome.
DNA microarrays are small pieces of glass or silicon that have many short pieces of DNA attached. Each short piece of DNA attached to a microarray is called a probe. Every probe is different and attaches specifically to one gene. Microarrays are often designed to contain enough probes to detect hundreds or thousands of different genes. If a microarray contains a probe for a specific gene, it can be used to check for the presence of that gene in a sample.
DNA microarray technology allows researchers to measure the amount of genes in a biological sample, such as cells or tissue. DNA microarrays allow researchers to simultaneously measure the amount of thousands of genes at once, which may greatly speed up experiments.
Researchers may use microarray technology in different types of studies. For example, some researchers may use microarrays to study cell development or to determined how one type of cell in the body is different from another. By identifying genes that are active at higher levels in one cell type than in another, researchers can learn more about how different types of cells develop and function in the body.
Researchers may also use microarrays to study disease. By comparing the amount of different RNAs in tissue from someone with a disease to the amount of RNAs in tissue from a healthy individual, researchers may be able to identify genes that are made at different levels in the sick individual and thereby identify genes that may be responsible for causing the disease.
For example, microarray technology has been used to study prostate cancer cells in order to identify specific genes that may be involved in prostate cancer progression. The levels of different RNAs from prostate cancer cells were compared to the levels of RNAs from normal cells using a DNA microarray. Using this approach, researchers were able to identify many new genes that may be involved in prostate cancer metastasis, or the process by which a tumor leaves its original site and invades other sites in the body.

Methods

Design microarray: DNA microarrays are small pieces of glass or silicon (slides) that have many short pieces of DNA attached. Each short piece of DNA attached to a microarray is called a probe. Every probe is different, and microarrays are often designed to contain probes that are able to detect hundreds or thousands of different genes. Probes on a microarray are arranged in rows and columns across the slide. They are placed in known locations, so that researchers can determine which probe is used to detect a specific gene. Probes are usually attached to the microarray slide with the help of robotic machines.
Microarrays that contain probes to detect thousands of human genes are manufactured and sold commercially. Researchers may also produce their own microarrays. This may be less expensive, especially if a researcher is interested in studying a smaller number of genes (such as 50-100) and needs a microarray with only enough probes to detect these genes.
Prepare sample: DNA microarrays are commonly used to detect the level of RNA in cells. However, they may also be used to detect the amount of DNA in cells. To perform a microarray experiment, DNA or RNA from cells or tissues of interest is obtained. This sample DNA or RNA is called the target. The DNA or RNA is then attached to a fluorescent dye. In some cases, researchers may study two different samples at once. In this type of experiment, two different colored dyes are used, and the DNA or RNA from each sample is attached to a different colored dye.
Hybridize the sample: The labeled target is then put onto the DNA microarray. If a probe designed to detect a specific piece of target DNA or RNA is present on the microarray, then that DNA or RNA will stick to the microarray. The more DNA or RNA present in the target sample, the more it will stick to the microarray. The process of letting the target stick to the probe is called hybridization. After the target is given a chance to stick to the microarray, the target that did not stick is washed off.
Detect sample: Researchers may measure how much of a specific RNA or DNA was present in the biological sample by measuring the amount of fluorescent signal produced by the target that sticks to the microarray. Each probe on a microarray is in a known location. If the probe for a certain gene has a strong fluorescent signal, it indicates that a large amount of RNA from that gene was present in the biological sample. If a certain probe produces a low (or no) fluorescent signal, it means that RNA from that gene was not present or was present in low amounts in the biological sample. The fluorescent signal is determined using a special machine called a plate reader that can detect and measure the strength of fluorescent light.
Because the probes on a microarray are organized in a pattern of rows and columns, the results from a microarray experiment typically look like a pattern of colored rows of dots. Dots with a very bright color correspond to probes that were able to detect the target, whereas dots without any color correspond to probes that did not detect the target in the sample. In some microarray experiments, two different target samples, each labeled with a different colored dye, are used. In these types of experiments, each sample may contain specific RNAs that hybridize to the same probe. The difference in the amount of RNA between the two samples can be determined by measuring the ratio of the two different dyes in a specific spot on the microarray.

Research

Study development: Microarray technology may be used by researchers to study how different cell types in an organism are formed, a process called development. For example, researchers have used microarray technology to study which genes are involved in making sperm in a type of worm called C. elegans. The researchers obtained RNA from worms that do produce sperm and RNA from worms that do not. The RNA from the sperm-producing worms was labeled with one dye, and the RNA from the non-sperm-producing worms was labeled with a different colored dye. The two different RNA samples were hybridized onto a microarray, and the researchers were able to determine which RNAs are more abundant in the worms that produce sperm than in the worms that do not produce sperm. Some of these RNAs may be involved in sperm production. By further studying the genes that correspond to these RNAs, researchers may be able to better understand the process through which sperm is produced.
DNA microarray technology has also been used to study heart development. A protein called the folate receptor, which functions in heart tissues to transport the B vitamin folate, is known to be important for heart development. Mice with mutations, or errors, in the folate receptor have developmental abnormalities in their hearts. In order to better understand how the folate receptor regulates heart development, researchers obtained RNA from healthy mice and RNA from mice with a folate receptor mutation. The RNA from healthy mice was labeled with one dye, and the RNA from mice with a folate receptor mutation was labeled with another. The two different RNA samples were hybridized onto a microarray, and the researchers were able to determine which RNAs were more abundant in the healthy mice than in the mutant mice. Differences in the expression of these genes can be further investigated by researchers, as they may be important for heart development.
Understand disease: Researchers may use microarray technology may to better understand human disease. In many human diseases, cells no longer carry out their normal function, and they may express specific genes more or less than normal. By measuring the levels of different RNAs in disease cells and comparing them to the levels of the same RNAs in healthy cells, researchers can learn which specific genes may be involved in a particular disease. Microarray technology is a powerful way to identify possible disease-causing genes because it allows researchers to check the levels of hundreds or thousands of genes in one experiment. This is important because a researcher often does not know which specific gene may be causing a disease and may therefore not know in advance which gene or genes to look for.
Microarray technology has been used to study prostate cancer cells in order to identify specific genes that may be involved in prostate cancer progression. The levels of different RNAs from prostate cancer cells were compared with the levels of RNAs from normal cells using a DNA microarray. Using this approach, researchers were able to identify many new genes that may be involved in prostate cancer metastasis, or the process by which a tumor leaves its original site and invades other sites in the body.
DNA microarrays have been used in a similar way to identify genes involved in other types of cancer, such as brain cancer, and in other diseases, such as Huntington's disease, a condition in which brain cells degenerate.

Implications

Diagnosing diseases: For many diseases, researchers have identified the genetic mutations responsible for causing the disease. If a patient displays signs of a particular genetic disease, DNA microarrays may be used to help diagnose the disease by detecting the abnormal DNA.
For example, mutations in clotting factor VIII are known to cause hemophilia, a disease in which blood does not clot properly. Microarray technology could be used to check for these mutations and to diagnose hemophilia. To perform these tests, DNA or RNA from patients could be hybridized to a customized DNA microarray that contains many short probes for the factor VIII gene. By checking for a probe to which the DNA or RNA from a patient does not hybridize strongly, researchers may be able to identify which part of the factor VIII gene contains a mutation. This is because a mutation in the factor VIII gene will cause the sequence of the gene to change, and it will not be able to bind as strongly to the probe, which was designed to detect the sequence of the normal gene.
Treating diseases: DNA microarray technology can be used to better understand some diseases. By understanding the specific changes in gene levels between an individual with a disease and in a healthy individual, researchers can better understand the disease mechanisms. This is because changes in the levels of genes can alter the function or behavior of cells, which may lead to a disease. By identifying the genes that become improperly regulated, scientists can better understand how the disease is caused. Using this information, they may be able to develop drugs to fight the disease. For example, researchers have found that some patients with breast cancer produce too much of a protein called HER2, and they have developed a drug that targets this protein.

Limitations

DNA microarrays may be used to detect the expression of hundreds or thousands of genes in just one experiment. However, in order to detect a specific gene, researchers need to have prior knowledge of the gene and its sequence in order to design a probe for that gene. Therefore, researchers cannot use microarrays to discover new genes. Instead, microarrays are used to detect genes that have already been characterized.

Future research

Not applicable.

Author information

This information has been edited and peer-reviewed by contributors to the Natural Standard Research Collaboration (www.naturalstandard.com).

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