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Gene expression analysis

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Also listed as: DNA expression
Related terms
Background
Methods
Research
Implications
Limitations
Safety
Future research
Author information
Bibliography

Related Terms
  • DNA expression, gene, hybridize, mircroarray, Northern blotting, probe, reverse transcription polymerase chain reaction, RT-PCR, target.

Background
  • Researchers use gene expression analysis to study how much of a particular gene is produced by a cell. Genes are considered the building blocks of cells because they provide instructions for making all proteins in the body. Although all cells in the human body contain the same DNA, cell types vary widely. For example, a skin cell is different from a brain cell in how it looks and what it does. Many of the differences among cells are not due to differences in the cellular DNA, but rather to differences in the amount and types of genes that the cell produces.
  • Cells produce genes by making RNA (ribonucleic acid) from DNA (deoxyribonucleic acid) and then converting the RNA into proteins. Each gene produces its own unique RNA. A gene is expressed if RNA for that gene is found in a cell. Measuring the amount of different RNAs in a cell can tell researchers how much of each gene is produced by that cell. A mutation is a change in the sequence of a gene, and it can affect how a protein functions.
  • DNA is located in a compartment of the cell called the nucleus and is packaged in structures called chromosomes. Human cells contain 46 chromosomes, including 22 pairs of autosomes and one pair of sex chromosomes (XX in females; XY in males), and each chromosome has hundreds of genes. Genes contain the instructions for making the proteins that perform all the functions in the human body. 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.
  • DNA contains four different chemical compounds called bases: cytosine, thymine, guanine, and adenine. In any given person, these bases are found in a particular order along the chromosomes, and it is the order of these bases that stores information for making genes. Even though the DNA sequences of different people are similar (on average, DNA is about 99.9% identical between two people), differences in DNA between people are important. Similar to DNA, RNA also contains chemical compounds called bases.
  • Researchers may use several different methods to study the expression of genes. Some methods, such as reverse transcription-polymerase chain reaction (RT-PCR) or Northern blotting, are best suited to study the expression of one or a few genes at a time. RT-PCR is a method that uses proteins called enzymes to amplify, or multiply, and detect genes, and Northern blotting is a method that allows researchers to separate and detect RNAs by size using a gel. Other methods, such as DNA microarray technology, allow researchers to measure the amount of hundreds or thousands of genes at the same time. DNA microarrays are small chips that have many probes, or short pieces of DNA, attached, which are able to detect a wide variety of RNAs in a sample.
  • Researchers may use gene expression analysis in different types of studies. For example, gene expression analysis may be used to study development or to find out how one type of cell in the body is different from another. By identifying genes that are present at higher levels in one cell type than in another, researchers can learn more about how different types of cells in the body develop and how they function. Researchers may also use gene expression analysis 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 play a role in disease development.

Methods
  • Researchers may use several different methods to study expression of genes. Some methods, such as reverse transcription-polymerase chain reaction (RT-PCR) and Northern blotting, are best suited to study the expression of one or a few genes at a time. Other methods, such as DNA microarray technology, allow researchers to measure the amount of hundreds or thousands of genes at the same time.
  • RT-PCR: Reverse transcription-polymerase chain reaction (RT-PCR) is a method of multiplying a piece of RNA millions of times, and it can be used to determine whether a specific RNA of interest is being expressed in a sample. To perform RT-PCR, RNA is first extracted from a biological sample, such as cells or a tissue. Then, a special type of protein, an enzyme called reverse transcriptase, is used to convert the RNA into DNA. Next, primers, or pieces of DNA that can specifically recognize the DNA of interest, are added to the sample. Researchers commonly select two different primers when performing RT-PCR, and these two primers typically are located at each end of the DNA sequence the researcher is interested in studying.
  • Next, another protein, DNA polymerase, is added to the sample. DNA polymerases are enzymes that are able to synthesize DNA, and in an RT-PCR reaction, the DNA polymerase makes thousands or millions of copies of the DNA sequence located between the two primers. Primers essentially define where the copy should start and end. However, a particular sequence will be amplified or copied only if it is present in the original sample. Therefore, by selecting primers for specific genes, researchers can determine whether a gene is expressed in a sample.
  • Northern blotting: Northern blotting is a method of detecting genes based on their size and charge. First, RNA is extracted from a biological sample, such as cells or a tissue. Next, this RNA is placed in a gel. A charge is applied to the gel, which causes the RNA to move at different speeds based on its charge and size. Larger RNA fragments will move more slowly, and smaller fragments will move more rapidly. After several hours, the different RNAs in the sample will become separated in the gel. The RNAs in the gel are then transferred onto a membrane.
  • Researchers may then use a probe, which is a sequence of DNA that can recognize and bind to the RNA of interest, in order to determine whether a specific RNA was present in the sample. Probes used for Northern blotting may be labeled with radioactive isotopes, which are compounds that emit radioactive particles. The presence of a target mRNA can be observed by exposing the membrane with a bound radioactive probe to a film that detects radiation. The presence of a specific mRNA will appear as a dark band on the film.
  • DNA microarray: DNA microarrays are small pieces of glass or silicon that have many short pieces of DNA attached. Each microarray has multiple pieces of DNA attached, and each short piece of attached DNA is called a probe. Every probe is different, and 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, then it can be used to check for the presence of that gene in a sample.
  • To detect the level of gene expression in a biological sample using a microarray, RNA from the sample attached to a fluorescent dye. This fluorescently labeled RNA is then placed onto the DNA microarray. If a probe designed to detect a specific RNA is present on the microarray, then that RNA will stick to the microarray. Genes that are produced at high levels will have more RNA in the sample, so more RNA from those genes will stick to the probes on the microarray than from genes produced at lower levels.
  • After the RNA is given a chance to stick to the microarray in a process called hybridization, all of the RNA that did not stick is washed off. Researchers may then measure how much RNA from a specific gene was present in the biological sample by measuring the amount of fluorescent signal that was produced by the microarray. Each probe on a microarray is contained in a known location. If the probe for a certain gene has a strong fluorescent signal, it means that a large amount of RNA from that gene was present in the cell. If a certain probe is producing a low fluorescent signal or none at all, it means that RNA from that gene was not present or was present in low amounts in the biological sample.

Research
  • Study development: Researchers may use gene expression analysis 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 produce sperm and some from worms that do not produce sperm. 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 RNA samples were hybridized onto a microarray, and the researchers were able to determine which RNAs were 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 by which sperm is produced in worms.
  • Northern blotting has been used by researchers to study brain development in monkeys. Researchers performed Northern blotting analysis to study the expression of a gene called neurogranin, which is known to be involved in brain cell function. The researchers found that neurogranin RNA was present in the brain at high levels at some times and at lower levels at other times in developing monkeys. This means that the developing brain needs neurogranin more at certain times than at other times. By further studying the periods during monkey development when neurogranin is most highly expressed, researchers may be able to learn more about how this gene is involved in regulating brain development.
  • Understand disease: Gene expression analysis may be used to better understand human diseases. In many human diseases, cells lose the ability to function normally and may make more or less of specific genes. By measuring the levels of different RNAs in disease cells and comparing them to the levels of the same RNAs in healthy cells, researchers may learn what specific genes are 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 researchers often do not know which specific gene may be causing a disease and may not know in advance which gene or genes to be looking 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 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. Metastasis is a process during 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 and lose their ability to function.

Implications
  • Diagnosing disease: For many diseases, researchers have identified genetic mutations that are responsible for causing the disease. If a patient displays symptoms of a particular genetic disease, gene expression analysis may be used to help make a diagnosis.
  • For example, mutations in clotting factor VIII are known to cause hemophilia, a disease in which blood does not clot properly, causing patients to bleed excessively. 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 where 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 a normal gene.
  • Gene expression analysis may be used to diagnose viral infections. Viruses have their own unique RNAs, so by checking for the presence of a viral RNA in a patient, doctors may determine whether that individual has been infected with a virus. For example, RT-PCR using primers specific for RNA of the hepatitis C virus (HCV) has been used to detect HCV in the blood of infected individuals because it can detect very low amounts of virus in the blood.
  • Fighting disease: Gene expression analysis can be used to better understand some diseases by giving researchers a tool to explore specific changes in gene levels between an individual with a disease and a healthy individual. This is because changes in the levels of genes may alter the function of cells or how the cells behave and may lead to diseases. By identifying the genes that become deregulated, scientists can better understand how the disease is caused and may be able to use this information 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 have developed a drug that targets this protein.

Limitations
  • Although gene expression analysis can tell a researcher whether a gene is actively being made, they cannot determine the function of the gene. Once researchers have found a specific gene that is expressed during a certain period in development or that is abnormally expressed in a certain disease, they may need to perform additional experiments to better understand how that gene functions.
  • Researchers must also be careful when making probes for detecting gene expression. A probe is a sequence of DNA or RNA that can recognize and bind to the gene of interest. Some regions on one gene may be very similar to regions on a different gene. Therefore, researchers may need to generate a probe that is specific to only the gene they are interested in, but is not similar to other genes. Otherwise, researchers may think they have identified the gene they are interested in when they have really identified a different but related gene.

Safety




Future research
  • Because humans have thousands of genes, gene expression analysis is an ongoing subject of research. Each year, researchers make new discoveries of specific genes that are expressed in developmental processes or that are improperly expressed in disease. The continued study of these genes will give researchers more insight into the developmental processes or diseases they have been linked to. For example, gene expression analysis may be used in the future to better understand cancer.

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

Bibliography
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