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In situ hybridization (ISH)


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Related Terms
  • Amplification, cancer, chromosome, colcemid, cytogenetic technique, deletion, disease, DNA, FISH, fluorescence, ISH karyotype, microscope, mRNA, probe, RNA.

  • In situ hybridization (ISH) is a method that researchers may use to determine whether a specific part of a chromosome or a specific messenger RNA (mRNA) is present in a cell. Chromosomes are located in a compartment of the cell called the nucleus and are composed of deoxyribonucleic acid (DNA) and proteins. Human cells contain 46 chromosomes, including 22 pairs of autosomes and one pair of sex chromosomes, and each chromosome has hundreds of genes. Genes contain the instructions for making the proteins that do the work in the human body. Messenger ribonucleic acids (mRNAs) are molecules that are made from DNA and are used by the cell to produce proteins.
  • To perform ISH, researchers may first identify a specific region of a chromosome or the mRNA for a specific gene that they are interested in studying. They then generate a probe, which is a sequence of DNA or RNA that can recognize and bind to the chromosomal region or mRNA of interest. ISH that is used to detect DNA or a chromosome has the potential to provide evidence that a gene is present in a cell. Likewise, ISH that is used to detect mRNA also has the potential to provide evidence that a gene is actively being made in a certain cell.
  • A label is a type of molecule that researchers attach to a probe so they can determine whether the probe has identified the target DNA or mRNA. There are several different ways that a probe used for ISH can be labeled. Some probes are labeled, with a fluorescent substance so they emit a colored light if the target of interest is present in a cell. This procedure may be called fluorescent in situ hybridization, or FISH. Some probes are designed to generate a colored stain. Probes that produce a colored stain may be observed using a normal light microscope, whereas probes that give off fluorescent light need to be detected with a more expensive fluorescent microscope.
  • In some diseases, such as cancer, genetic mutations occur that can cause part of a chromosome to be deleted, repeated, or reversed in orientation. Also, in some diseases, specific mRNAs may be produced at higher or lower levels. By observing the differences in how chromosomes or mRNAs from different cells stain with a probe, a researcher may gain further insight into the genetic mutations that cause a particular disease.

  • To perform in situ hybridization (ISH), researchers typically need to carry out several steps.
  • Obtain cells: First, a line of cells is grown in the laboratory, for example, in a cell culture dish. Cells lines made from blood, amniotic fluid, or bone marrow are commonly used for analysis. After the cells have multiplied, a chemical such as colcemid is used to stop the cells from growing further. Colcemid inhibits the growth of cells when they reach a stage of the cell cycle called metaphase, which is when the chromosomes are easily visible.
  • Prepare cells for observation: Once the cells have been obtained, they are exposed to a hypotonic solution, which has a relatively low concentration of dissolved molecules. This solution expands the cells and spreads the chromosomes out so that they are easier to examine. The cells are killed using a fixative, such as methanol and acetic acid, and then transferred to a microscope where the chromosomes can be observed. Some of the metaphase cells are dropped onto a microscope slide, which is a thin piece of glass, and then spread apart on the slide with a small-tipped instrument. They are then dried so they become attached to a cell.
  • Using tissues: When performing ISH, researchers may choose to study part of a tissue, such as the heart or kidney, rather than just individual cells. To study a tissue, researchers typically first use a fixative, such as paraformaldehyde, to preserve the sample. The fixed tissues are then sliced into thin strips called sections.
  • Obtain probe: To perform ISH, researchers may first identify a region of a chromosome or the mRNA for a specific gene that they are interested in studying. They then generate a probe, which is a sequence of DNA or RNA that can recognize and bind to the chromosomal region or mRNA of interest.
  • Researchers can obtain the DNA material for a probe in several different ways. For example, they may perform a technique called chromosome microdissection, in which they physically remove part of a chromosome using a fine, sharp glass needle, and then use it as a probe. Or they may obtain a piece of DNA to use as a probe from a DNA library, which is a large collection of isolated and purified DNA fragments frequently used in DNA sequencing projects. DNA libraries may be purchased from biotechnology companies.
  • To generate an RNA probe, researchers typically use proteins called RNA polymerases. RNA polymerases are enzymes that a cell uses to produce mRNA from DNA. Researchers may use these enzymes in the laboratory to produce RNA probes from a selected piece of DNA. RNA probes are not as easy to work with as DNA probes because they are less stable and degrade more easily. However, RNA probes are also better at detecting mRNA than DNA probes.
  • Apply probe to cells: Using the probe, researchers can determine whether the cells or tissue sections they are studying contain the region of DNA or the mRNA that the probe was designed to detect. When detecting DNA, the cells, which have been attached to microscope slides, are heated so that the DNA will be more accessible to the probe. The probe is then added to the slide or the tissue section. If the chromosomal region or mRNA of interest is present, the probe will bind to the chromosome or mRNA. After the probe has been given time to bind to the DNA or mRNA, which may take several days, the cells or tissue section are washed so that the probe remains only in samples in which it has detected and bound to a target.
  • In some types of ISH, the probes are labeled with a fluorescent substance so they emit a colored light when viewed with a fluorescent microscope if the chromosomal region or mRNA of interest is present. This type of ISH is called fluorescent in situ hybridization or FISH. When observed under the microscope, FISH probes that are bound to a target often appear as small colored dots. In some cases, researchers may choose to use more than one probe at the same time in a FISH experiment. Different colored FISH probes are available so that in an experiment one probe could appear as a red dot and another as a blue dot.
  • In some types of ISH, the probes that researchers use are designed to generate a colored stain. If a target chromosomal region or mRNA is present in a cell, researchers will be able to observe the stain by looking at a cellular sample under a light microscope. To generate the colored stain, an enzyme, a type of protein that helps carry out a chemical reaction, is linked to the probe. When a chemical is then added to the cellular sample, the enzyme carries out a reaction on the chemical, causing a colored stain to form. These colored stains can be observed by researchers using a normal light microscope. Commonly, an enzyme called peroxidase and a chemical called diaminobenzidine are used, which results in the formation of a dark brown color.

  • Using in situ hybridization (ISH), researchers can check for differences in the chromosomes between two different cells or for differences in the amount of mRNA between two different samples.
  • In some cases, ISH may be able to demonstrate that one cell has an extra copy of a specific chromosome. In other cases, ISH may be able to detect smaller variations in a particular chromosome, such as a repeated region or a deletion in the chromosome. ISH is often used to study different types of diseases, such as cancer, in which mutations occur that cause extra copies of chromosomes to be present or that cause a portion of a chromosome to become deleted, repeated, or reversed in orientation.
  • ISH may be used to study evolutionary relationships between different species. If a probe is designed to detect a specific chromosomal region in one species, that probe can also be used to check for the same chromosomal region in another species. If the species are closely related, the probe will also bind to the chromosome of the second species. If the species are distantly related, however, the probe will no longer be able to bind to the chromosome of the second species. ISH may also be used to look for other changes that occur between two species during evolution, such as amplification of a particular chromosomal region. Amplification is a type of mutation in which a region of DNA becomes repeated one or more times.
  • In some cases, ISH may not be used to detect a region on a chromosome but is instead used to check for the presence of messenger RNA (mRNA). MRNA is a molecule made from DNA and is used by a cell to produce proteins. ISH that is used to detect DNA or a chromosome may tell a researcher whether a gene is present in a cell, and ISH that is used to detect mRNA may tell a researcher whether a gene is actively being made in a cell. Researchers commonly use ISH to detect RNA when studying the development of an organism. In this context, RNA ISH may be used to determine which specific genes are active and functioning during the development of an organism.

  • Diagnosis: In situ hybridization (ISH) can be used to diagnose certain human diseases prenatally, before a baby is born. For example, diagnosis of genetic diseases such as trisomy 18 or Down syndrome (also known as trisomy 21) may be performed on a developing fetus through amniocentesis, in which the amniotic fluid surrounding the fetus is sampled through a needle. Cells in the amniotic fluid can be used to perform ISH and to check for an extra copy of chromosome 18 (in the case of trisomy 18) or an extra copy of chromosome 21 (in the case of Down syndrome). Trisomy 18 is a genetic condition that leads to a wide range of physical and mental developmental defects, including heart defects, kidney defects, intellectual disability, and feeding problems. Down syndrome is a genetic disorder that is characterized by cognitive dysfunction that ranges from mild to severe.
  • ISH may also be used by doctors to diagnose diseases in children or adults. For example, Prader-Willi syndrome is a genetic disease in which part of chromosome 15 is deleted. Using ISH, doctors can design probes for chromosome 15 and detect the chromosome 15 deletion in patients. ISH may also be used to detect genetic diseases in which part of a particular chromosome has undergone an expansion. For example, in the neuromuscular disease myotonic dystrophy, part of chromosome 3 is duplicated many times.
  • Bacterial detection: Although in many cases ISH is used to analyze a cultured line of cells, ISH can also be used directly on nondividing bacterial cells to detect a bacterial infection. By generating probes that are specific for different strains of bacteria, doctors can detect whether that strain of bacteria is present in a patient's blood. For example, ISH has been used to detect the bacterial pathogen Pseudomonas in blood samples. Pseudomonas is a common cause of urinary tract infections.
  • Studying development: Researchers may use ISH to determine which specific mRNAs are actively made at a certain stage in development. This may give researchers insight into which specific genes function in different developmental processes and which are involved in making an adult organism. For example, using RNA ISH, researchers have learned more about the specific genes involved in the development of teeth in mice. By performing RNA ISH, they identified some genes that are produced in developing teeth, and in which cells those genes are found.
  • Studying cancer: ISH may be used by doctors to make a prognosis for some types of cancer. Using ISH, researchers have found that in prostate cancer cells, a specific gene called the epidermal growth factor receptor is present in many more copies than in normal cells. Genetic changes have also been observed using ISH in other types of cancer, such as cervical and breast cancers. These genetic changes may be used by doctors to diagnose cancers or to make a prognosis for the disease.
  • Understanding diseases: ISH can be used to better understand some diseases. By understanding the specific rearrangements that chromosomes have undergone in patients, researchers can better understand the disease. This is because rearrangements may cause specific genes to become deleted or amplified, and the genes that have increased or decreased in number may play a role in causing the disease. By identifying these genes, scientists can better understand how the disease is caused, and may be able to use this information to develop drugs to fight it. This approach is useful for the study of some types of cancer, such as lung cancer, in which chromosomes become mutated and undergo rearrangements.

  • One drawback of in situ hybridization (ISH) is that in order to generate a probe to detect a specific region of a chromosome or a specific mRNA, some information about the genetic sequence of that chromosome or gene must already be known. When studying a chromosome, it is not always necessary to know the specific DNA sequence of the area that is being examined, and researchers sometimes generate probes based on regions of chromosomes near the regions they are interested in studying.
  • It is vital for researchers to be extremely careful when generating a probe because certain regions on a specific chromosome or mRNA may appear very similar to regions on an entirely different chromosome or mRNA. Researchers must be aware of this possibility and generate a probe that is specific to a region of a chromosome and distinct from any other region.


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