thumb|Multiplex RNA visualization in cells using ViewRNA FISH Assays
right|thumb|A metaphase cell positive for the bcr/abl rearrangement (associated with [[chronic myelogenous leukemia) using FISH. The chromosomes can be seen in blue. The chromosome that is labeled with green and red spots (upper left) is the one where the rearrangement is present.]]
Fluorescence in situ hybridization (FISH) is a molecular cytogenetic technique that uses fluorescent probes that bind to specific parts of a nucleic acid sequence with a high degree of sequence complementarity. It was developed by biomedical researchers in the early 1980s to detect and localize the presence or absence of specific DNA sequences on chromosomes. Fluorescence microscopy can be used to determine where the fluorescent probe is bound to the chromosomes. FISH is often used to find specific features in DNA for genetic counseling, medicine, and species identification.
FISH can also be used to detect and localize specific RNA targets (mRNA, lncRNA, and miRNA) in cells, circulating tumor cells, and tissue samples. In this context, it helps define the spatial and temporal patterns of gene expression within cells and tissues.
Probes – RNA and DNA
thumb|ViewRNA detection of miR-133(green) and myogenin mRNA (red) in C2C12 differentiating cells
In biology, a probe is a single strand of DNA or RNA that is complementary to a nucleotide sequence of interest.
RNA probes can be designed for any gene or any sequence within a gene for visualization of mRNA, lncRNA and miRNA in tissues and cells. FISH is used by examining the cellular reproduction cycle, specifically interphase of the nuclei for any chromosomal abnormalities. FISH allows the analysis of a large series of archival cases much easier to identify the pinpointed chromosome by creating a probe with an artificial chromosomal foundation that will attract similar chromosomes. The process is done in 3 main procedures: tissue preparation (pre-hybridization), hybridization, and washing (post-hybridization).
The tissue preparation starts by collecting the appropriate tissue sections to perform RNA FISH. First, cells, circulating tumor cells (CTCs), formalin-fixed paraffin-embedded (FFPE), or frozen tissue sections are "fixed." Fixing involves treating live cells with a chemical fixative like 4% formaldehyde or paraformaldehyde (PFA) in phosphate buffered saline (PBS). which is normally conducted with a detergent like triton-X, which is pertinent for cell staining as dead cells no longer have a functioning membrane. Cell permeabilization can also be done using a 70 percent Ethanol solution and over night refrigeration. After fixation, samples are permeabilized to allow the penetration of hybridization reagents. The use of detergents at a 0.1% concentration is commonly used to enhance the tissue permeability such as Tween-20 or Triton X-100.
It is critical for the hybridization process to have all optimal conditions to have a successful in situ result, including temperature, pH, salt concentration, and time of the hybridization reaction. After checking all the necessary conditions, hybridization steps can be started by first adding a target-specific probe, composed of 20 oligonucleotide pairs, hybridizes to the target RNA(s). Separate but compatible signal amplification systems enable the multiplex assay (up to two targets per assay). Signal amplification is achieved via series of sequential hybridization steps.
After the hybridization steps, washing steps are performed. These steps aim to remove nonspecific hybrids and get rid of unbound probe molecules from the samples to reduce any background signaling. The use of ethanol washes are typically used at this stage to reduce autofluorescence in tissues or cells. At the end of the assay the tissue samples are visualized under a fluorescence microscope such as the confocal fluorescence microscope and the Keyence microscope. or smFISH, is a method of detecting and quantifying mRNA and other long RNA molecules in a thin layer of tissue sample. Targets can be reliably imaged through the application of multiple short singly labeled oligonucleotide probes. The binding of up to 48 fluorescent labeled oligos to a single molecule of mRNA provides sufficient fluorescence to accurately detect and localize each target mRNA in a wide-field fluorescent microscopy image. Probes not binding to the intended sequence do not achieve sufficient localized fluorescence to be distinguished from background.
Single-molecule RNA FISH assays can be performed in simplex or multiplex, and can be used as a follow-up experiment to quantitative PCR, or imaged simultaneously with a fluorescent antibody assay. The technology has potential applications in cancer diagnosis, neuroscience, gene expression analysis, and companion diagnostics.
FIBRE FISH
In an alternative technique to interphase or metaphase preparations, fiber FISH, interphase chromosomes are attached to a slide in such a way that they are stretched out in a straight line, rather than being tightly coiled, as in conventional FISH, or adopting a chromosome territory conformation, as in interphase FISH. This is accomplished by applying mechanical shear along the length of the slide, either to cells that have been fixed to the slide and then lysed, or to a solution of purified DNA. A technique known as chromosome combing is increasingly used for this purpose. The extended conformation of the chromosomes allows dramatically higher resolution – even down to a few kilobases. The preparation of fiber FISH samples, although conceptually simple, is a rather skilled art, and only specialized laboratories use the technique routinely.
Q-FISH
Q-FISH combines FISH with PNAs and computer software to quantify fluorescence intensity. This technique is used routinely in telomere length research.
Flow-FISH
Flow-FISH uses flow cytometry to perform FISH automatically using per-cell fluorescence measurements.
MA-FISH
Microfluidics-assisted FISH (MA-FISH) uses a microfluidic flow to increase DNA hybridization efficiency, decreasing expensive FISH probe consumption and reduce the hybridization time. MA-FISH is applied for detecting the HER2 gene in breast cancer tissues.
MAR-FISH
Microautoradiography FISH is a technique to combine radio-labeled substrates with conventional FISH to detect phylogenetic groups and metabolic activities simultaneously.
Hybrid Fusion-FISH
Hybrid Fusion FISH (HF-FISH) uses primary additive excitation/emission combination of fluorophores to generate additional spectra through a labeling process known as dynamic optical transmission (DOT). Three primary fluorophores are able to generate a total of 7 readily detectable emission spectra as a result of combinatorial labeling using DOT. Hybrid Fusion FISH enables highly multiplexed FISH applications that are targeted within clinical oncology panels. The technology offers faster scoring with efficient probesets that can be readily detected with traditional fluorescent microscopes.
MERFISH
Multiplexed error-robust fluorescence in situ hybridization is a highly multiplexed version of smFISH. It uses combinatorial labeling, followed by imaging, and then error-resistant encoding
Medical applications
Often parents of children with a developmental disability want to know more about their child's conditions before choosing to have another child. These concerns can be addressed by analysis of the parents' and child's DNA. In cases where the child's developmental disability is not understood, the cause of it can potentially be determined using FISH and cytogenetic techniques. Examples of diseases that are diagnosed using FISH include Prader-Willi syndrome, Angelman syndrome, 22q13 deletion syndrome, chronic myelogenous leukemia, acute lymphoblastic leukemia, Cri-du-chat, Velocardiofacial syndrome, and Down syndrome. FISH on sperm cells is indicated for men with an abnormal somatic or meiotic karyotype as well as those with oligozoospermia, since approximately 50% of oligozoospermic men have an increased rate of sperm chromosome abnormalities. The analysis of chromosomes 21, X, and Y is enough to identify oligozoospermic individuals at risk.
alt= This figure outlines the process of fluorescent in situ hybridization (FISH) used for pathogen identification. First, a sample of the infected tissue is taken from the patient. Then an oligonucleotide that is complementary to the suspected pathogen's genetic code is synthesized and chemically tagged with a fluorescent probe. The collected tissue sample must then be chemically treated in order to make the cell membranes permeable to the fluorescently tagged oligonucleotide. After the tissue sample is treated, the tagged complementary oligonucleotide is added. The fluorescently tagged oligonucleotide will only bind to the complementary DNA of the suspected pathogen. If the pathogen is present in the tissue sample, then the pathogen's cells will glow/fluoresce after treatment with the tagged oligonucleotide. All other cells will not glow after treatment.|thumb|General process of fluorescent in situ hybridization (FISH) used for bacterial pathogen identification. First, an infected tissue sample is taken from the patient. Then an oligonucleotide complementary to the suspected pathogen's genetic code is chemically tagged with a fluorescent probe. The tissue sample is chemically treated in order to make the cell membranes permeable to the fluorescently tagged oligonucleotide. The fluorescent tag is then added and only binds to the complementary DNA of the suspected pathogen. If the pathogen is present in the tissue sample, then the pathogen's cells will fluoresce after treatment with the tagged oligonucleotide. No other cells will glow.|450x450px
Species identification
FISH has been extensively studied as a diagnostic technique for the identification of pathogens in the field of medical microbiology. Although it has been proven to be a useful and applicable technique, it is still not widely applied in diagnostic laboratories. The short time to diagnosis (less than 2 hours) has been a major advantage compared with biochemical differentiation, but this advantage is challenged by MALDI-TOF-MS which allows the identification of a wider range of pathogens compared with biochemical differentiation techniques. Using FISH for diagnostic purposes has found its purpose when immediate species identification is needed, specifically for the investigation of blood cultures for which FISH is a cheap and easy technique for preliminary rapid diagnosis.
Virtual karyotype
Virtual karyotyping is another cost-effective, clinically available alternative to FISH panels using thousands to millions of probes on a single array to detect copy number changes, genome-wide, at unprecedented resolution. Currently, this type of analysis will only detect gains and losses of chromosomal material and will not detect balanced rearrangements, such as translocations and inversions which are hallmark aberrations seen in many types of leukemia and lymphoma.
Spectral karyotype
Spectral karyotyping is an image of colored chromosomes. Spectral karyotyping involves FISH using multiple forms of many types of probes with the result to see each chromosome labeled through its metaphase stage. This type of karyotyping is used specifically when seeking out chromosome arrangements.
Chromosome evolution
thumb|Human chromosomes painted with DNA from mouse chromosome 11 showing hybridization signals on human chromosomes 17, 5, 2, 7, and 22 and some other chromosomes. That is, an ancestral chromosome broke up into multiple fragments that can still be found in many human chromosomes.
FISH can be used to study the evolution of chromosomes. Species that are related have similar chromosomes. This homology can be detected by gene or genome sequencing but also by FISH. For instance, human and chimpanzee chromosomes are very similar, and FISH can demonstrate that the common ancestor of chimpanzees and humans had two smaller chromosomes, and in the human lineage these fused to result in one human chromosome. Similarly, species that are more distantly related, have similar chromosomes but with increasing genetic distance, chromosomes tend to break and fuse and thus result in mosaic chromosomes. This can be impressively demonstrated by FISH (see figure).