View source for Fluorescence in situ hybridization

{{Short description|Genetic testing technique}}
{{DISPLAYTITLE:Fluorescence ''in situ'' hybridization}}
[[File:Multiplex ViewRNA FISH Assay in Jurkat and HeLa cells.jpg|thumb|Multiplex RNA visualization in cells using ViewRNA FISH Assays]]
[[File:Bcrablmet.jpg|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 [[cytogenetics|molecular cytogenetic]] technique that uses [[hybridization probe|fluorescent probes]] that bind to only particular parts of a [[nucleic acid]] sequence with a high degree of sequence [[Complementarity (molecular biology)|complementarity]]. It was developed by biomedical researchers in the early 1980s<ref>{{cite journal | vauthors = Langer-Safer PR, Levine M, Ward DC | title = Immunological method for mapping genes on drosophila polytene chromosomes | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 79 | issue = 14 | pages = 4381–4385 | date = July 1982 | pmid = 6812046 | pmc = 346675 | doi = 10.1073/pnas.79.14.4381 | doi-access = free | bibcode = 1982PNAS...79.4381L }}</ref> to detect and localize the presence or absence of specific [[DNA]] [[DNA sequence|sequences]] on [[chromosome]]s. [[Fluorescence microscopy]] can be used to find out where the fluorescent probe is bound to the chromosomes. FISH is often used for finding specific features in DNA for use in [[genetic counseling]], medicine, and species identification.<ref>{{cite journal | vauthors = Amann R, Fuchs BM | title = Single-cell identification in microbial communities by improved fluorescence in situ hybridization techniques | journal = Nature Reviews. Microbiology | volume = 6 | issue = 5 | pages = 339–348 | date = May 2008 | pmid = 18414500 | doi = 10.1038/nrmicro1888 | s2cid = 22498325 }}</ref> FISH can also be used to detect and localize specific RNA targets ([[mRNA]], [[Long non-coding RNA|lncRNA]] and [[miRNA]]){{Citation needed|date=November 2020}} in cells, circulating tumor cells, and tissue samples. In this context, it can help define the spatial-temporal patterns of [[gene expression]] within cells and tissues.
== Probes – RNA and DNA ==
[[File:MicroRNA and mRNA visualization in differentiating C1C12 cells.jpg|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 [[Messenger RNA|mRNA]],<ref>{{cite journal | vauthors = Anthony SJ, St Leger JA, Pugliares K, Ip HS, Chan JM, Carpenter ZW, Navarrete-Macias I, Sanchez-Leon M, Saliki JT, Pedersen J, Karesh W, Daszak P, Rabadan R, Rowles T, Lipkin WI | display-authors = 6 | title = Emergence of fatal avian influenza in New England harbor seals | journal = mBio | volume = 3 | issue = 4 | pages = e00166–e00112 | year = 2012 | pmid = 22851656 | pmc = 3419516 | doi = 10.1128/mBio.00166-12 }}</ref><ref>{{cite journal | vauthors = Everitt AR, Clare S, Pertel T, John SP, Wash RS, Smith SE, Chin CR, Feeley EM, Sims JS, Adams DJ, Wise HM, Kane L, Goulding D, Digard P, Anttila V, Baillie JK, Walsh TS, Hume DA, Palotie A, Xue Y, Colonna V, Tyler-Smith C, Dunning J, Gordon SB, Smyth RL, Openshaw PJ, Dougan G, Brass AL, Kellam P | display-authors = 6 | title = IFITM3 restricts the morbidity and mortality associated with influenza | journal = Nature | volume = 484 | issue = 7395 | pages = 519–523 | date = March 2012 | pmid = 22446628 | pmc = 3648786 | doi = 10.1038/nature10921 | bibcode = 2012Natur.484..519. }}</ref><ref>{{cite journal | vauthors = Louzada S, Adega F, Chaves R | title = Defining the sister rat mammary tumor cell lines HH-16 cl.2/1 and HH-16.cl.4 as an in vitro cell model for Erbb2 | journal = PLOS ONE | volume = 7 | issue = 1 | pages = e29923 | year = 2012 | pmid = 22253826 | pmc = 3254647 | doi = 10.1371/journal.pone.0029923 | doi-access = free | bibcode = 2012PLoSO...729923L }}</ref> [[Long non-coding RNA|lncRNA]]<ref name="pmid21233348">{{cite journal | vauthors = Ting DT, Lipson D, Paul S, Brannigan BW, Akhavanfard S, Coffman EJ, Contino G, Deshpande V, Iafrate AJ, Letovsky S, Rivera MN, Bardeesy N, Maheswaran S, Haber DA | display-authors = 6 | title = Aberrant overexpression of satellite repeats in pancreatic and other epithelial cancers | journal = Science | volume = 331 | issue = 6017 | pages = 593–596 | date = February 2011 | pmid = 21233348 | pmc = 3701432 | doi = 10.1126/science.1200801 | bibcode = 2011Sci...331..593T }}</ref><ref>{{cite journal | vauthors = Zhang B, Arun G, Mao YS, Lazar Z, Hung G, Bhattacharjee G, Xiao X, Booth CJ, Wu J, Zhang C, Spector DL | display-authors = 6 | title = The lncRNA Malat1 is dispensable for mouse development but its transcription plays a cis-regulatory role in the adult | journal = Cell Reports | volume = 2 | issue = 1 | pages = 111–123 | date = July 2012 | pmid = 22840402 | pmc = 3408587 | doi = 10.1016/j.celrep.2012.06.003 }}</ref><ref>{{cite journal | vauthors = Lee K, Kunkeaw N, Jeon SH, Lee I, Johnson BH, Kang GY, Bang JY, Park HS, Leelayuwat C, Lee YS | display-authors = 6 | title = Precursor miR-886, a novel noncoding RNA repressed in cancer, associates with PKR and modulates its activity | journal = RNA | volume = 17 | issue = 6 | pages = 1076–1089 | date = June 2011 | pmid = 21518807 | pmc = 3096040 | doi = 10.1261/rna.2701111 }}</ref> and [[miRNA]] in tissues and cells. FISH is used by examining the cellular reproduction cycle, specifically interphase of the nuclei for any chromosomal abnormalities.<ref name="pmid18234275">{{cite journal | vauthors = Bernasconi B, Karamitopoulou-Diamantis E, Karamitopolou-Diamantiis E, Tornillo L, Lugli A, Di Vizio D, Dirnhofer S, Wengmann S, Glatz-Krieger K, Fend F, Capella C, Insabato L, Terracciano LM | display-authors = 6 | title = Chromosomal instability in gastric mucosa-associated lymphoid tissue lymphomas: a fluorescent in situ hybridization study using a tissue microarray approach | journal = Human Pathology | volume = 39 | issue = 4 | pages = 536–542 | date = April 2008 | pmid = 18234275 | doi = 10.1016/j.humpath.2007.08.009 }}</ref> 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.<ref name="pmid18234275" /> The hybridization signals for each probe when a nucleic abnormality is detected.<ref name="pmid18234275" /> Each probe for the detection of mRNA and lncRNA is composed of ~20-50 oligonucleotide pairs, each pair covering a space of 40–50 bp. The specifics depend on the specific FISH technique used. For miRNA detection, the probes use proprietary chemistry for specific detection of miRNA and cover the entire miRNA sequence.
[[File:Urovysion on Duet.png|thumb|right|Urothelial cells marked with four different probes]]
Probes are often derived from fragments of DNA that were isolated, purified, and amplified for use in the [[Human Genome Project]].  The size of the human genome is so large, compared to the length that could be sequenced directly, that it was necessary to divide the genome into fragments. (In the eventual analysis, these fragments were put into order by digesting a copy of each fragment into still smaller fragments using sequence-specific endonucleases, measuring the size of each small fragment using [[size exclusion chromatography|size-exclusion chromatography]], and using that information to determine where the large fragments overlapped one another.) To preserve the fragments with their individual DNA sequences, the fragments were added into a system of continually replicating bacteria populations.  Clonal populations of bacteria, each population maintaining a single artificial chromosome, are stored in various laboratories around the world.  The artificial chromosomes ([[bacterial artificial chromosome|BAC]]) can be grown, extracted, and labeled, in any lab containing a library. Genomic libraries are often named after the institution in which they were developed. An example being the RPCI-11 library, which is named after [[Roswell Park Comprehensive Cancer Center]] (formerly known as Roswell Park Cancer Institute) in [[Buffalo, New York]]. These fragments are on the order of 100 thousand base-pairs, and are the basis for most FISH probes.
=== Preparation and hybridization process – RNA ===
The purpose of using RNA FISH is to detect target mRNA transcripts in cells, tissue sections, or even whole-mounts.<ref name="Young e8806">{{cite journal | vauthors = Young AP, Jackson DJ, Wyeth RC | title = A technical review and guide to RNA fluorescence in situ hybridization | journal = PeerJ | volume = 8 | pages = e8806 | date = 2020-03-19 | pmid = 32219032 | pmc = 7085896 | doi = 10.7717/peerj.8806 | doi-access = free }}</ref> 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 cell]]s (CTCs), formalin-fixed paraffin-embedded (FFPE), or frozen tissue sections are fixed. Some commonly used fixatives are 4% formaldehyde or paraformaldehyde (PFA) in phosphate buffered saline (PBS).<ref name="Young e8806"/> FISH has also been successfully done on unfixed cells.<ref>{{cite book | vauthors = Haroon MF, Skennerton CT, Steen JA, Lachner N, Hugenholtz P, Tyson GW | title = Microbial Metagenomics, Metatranscriptomics, and Metaproteomics | chapter = In-solution fluorescence in situ hybridization and fluorescence-activated cell sorting for single cell and population genome recovery | series = Methods in Enzymology | volume = 531 | pages = 3–19 | date = 2013 | pmid = 24060113 | doi = 10.1016/B978-0-12-407863-5.00001-0 | isbn = 9780124078635 }}</ref> 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.<ref name="Cui 2016 89">{{cite journal | vauthors = Cui C, Shu W, Li P | title = Fluorescence In situ Hybridization: Cell-Based Genetic Diagnostic and Research Applications | journal = Frontiers in Cell and Developmental Biology | volume = 4 | pages = 89 | date = 2016 | pmid = 27656642 | pmc = 5011256 | doi = 10.3389/fcell.2016.00089 | doi-access = free }}</ref>
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.<ref>{{cite journal | vauthors = Xie F, Timme KA, Wood JR | title = Using Single Molecule mRNA Fluorescent in Situ Hybridization (RNA-FISH) to Quantify mRNAs in Individual Murine Oocytes and Embryos | journal = Scientific Reports | volume = 8 | issue = 1 | pages = 7930 | date = May 2018 | pmid = 29785002 | pmc = 5962540 | doi = 10.1038/s41598-018-26345-0 | bibcode = 2018NatSR...8.7930X }}</ref>
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.<ref>{{cite journal | vauthors = Oliveira VC, Carrara RC, Simoes DL, Saggioro FP, Carlotti CG, Covas DT, Neder L | title = Sudan Black B treatment reduces autofluorescence and improves resolution of in situ hybridization specific fluorescent signals of brain sections | journal = Histology and Histopathology | volume = 25 | issue = 8 | pages = 1017–1024 | date = August 2010 | pmid = 20552552 | doi = 10.14670/HH-25.1017 }}</ref> 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.<ref name="Cui 2016 89"/>
=== Preparation and hybridization process – DNA ===
[[File:FISH (Fluorescent In Situ Hybridization).jpg|thumb|right|Scheme of the principle of the FISH Experiment to localize a gene in the nucleus.]]
First, a probe is constructed. The probe must be large enough to hybridize specifically with its target but not so large as to impede the hybridization process.  The probe is [[Fluorescent tag|tagged]] directly with [[fluorophore]]s, with targets for [[antibodies]] or with [[biotin]]. Tagging can be done in various ways, such as [[nick translation]], or [[polymerase chain reaction]] using tagged  [[nucleotide]]s.
Then, an [[interphase]] or [[metaphase]] chromosome preparation is produced. The chromosomes are firmly attached to a [[Substrate (biochemistry)|substrate]], usually glass. Repetitive DNA sequences must be blocked by adding short fragments of DNA to the sample. The probe is then applied to the chromosome DNA and incubated for approximately 12 hours while hybridizing. Several wash steps remove all unhybridized or partially hybridized probes.  The results are then visualized and quantified using a microscope that is capable of exciting the dye and recording images.
If the fluorescent signal is weak, amplification of the signal may be necessary in order to exceed the detection threshold of the [[microscope]]. Fluorescent signal strength depends on many factors such as probe labeling efficiency, the type of probe, and the type of dye. Fluorescently tagged [[antibodies]] or [[streptavidin]] are bound to the dye molecule.  These secondary components are selected so that they have a strong signal.
== Variations on probes and analysis ==
FISH is a very general technique.  The differences between the various FISH techniques are usually due to variations in the sequence and labeling of the probes; and how they are used in combination.  Probes are divided into two generic categories: cellular and acellular. In fluorescent "in situ" hybridization refers to the cellular placement of the probe
Probe size is important because shorter probes hybridize less specifically than longer probes, so that long enough strands of DNA or RNA (often 10–25 nucleotides) which are complementary to a given target sequence are often used to locate a target. The overlap defines the resolution of detectable features. For example, if the goal of an experiment is to detect the breakpoint of a [[Chromosomal translocation|translocation]], then the overlap of the probes — the degree to which one DNA sequence is contained in the adjacent probes — defines the minimum window in which the breakpoint may be detected.
The mixture of probe sequences determines the type of feature the probe can detect.  Probes that hybridize along an entire chromosome are used to count the number of a certain chromosome, show translocations, or identify extra-chromosomal fragments of [[chromatin]]. This is often called "whole-chromosome painting." If every possible probe is used, every chromosome, (the whole genome) would be marked fluorescently, which would not be particularly useful for determining features of individual sequences.   However, it is possible to create a mixture of smaller probes that are specific to a particular region (locus) of DNA; these mixtures are used to detect [[Genetic deletion|deletion mutations]].  When combined with a specific color, a locus-specific probe mixture is used to detect very specific translocations.  Special locus-specific probe mixtures are often used to count chromosomes, by binding to the [[centromeric]] regions of chromosomes, which are distinctive enough to identify each chromosome (with the exception of [[Chromosome 13]], [[Chromosome 14|14]], [[Chromosome 21|21]], [[Chromosome 22|22]].)
A variety of other techniques uses mixtures of differently colored probes. A range of colors in mixtures of fluorescent dyes can be detected, so each human chromosome can be identified by a characteristic color using whole-chromosome probe mixtures and a variety of ratios of colors.  Although there are more chromosomes than easily distinguishable fluorescent dye colors, ratios of probe mixtures can be used to create ''secondary'' colors.  Similar to [[comparative genomic hybridization]], the probe mixture for the secondary colors is created by mixing the correct ratio of two sets of differently colored probes for the same chromosome.  This technique is sometimes called M-FISH.
The same physics that make a variety of colors possible for M-FISH can be used for the detection of translocations.  That is, colors that are adjacent appear to overlap; a secondary color is observed.  Some assays are designed so that the secondary color will be present or absent in cases of interest.  An example is the detection of [[Philadelphia chromosome|BCR/ABL]] translocations, where the secondary color indicates disease.   This variation is often called double-fusion FISH or D-FISH.  In the opposite situation—where the absence of the secondary color is pathological—is illustrated by an assay used to investigate translocations where only one of the breakpoints is known or constant.  Locus-specific probes are made for one side of the breakpoint and the other intact chromosome.  In normal cells, the secondary color is observed, but only the primary colors are observed when the translocation occurs.  This technique is sometimes called "break-apart FISH".
=== Single-molecule RNA FISH ===
Single-molecule RNA FISH, also known as Stellaris® RNA FISH<ref>{{cite book | vauthors = Orjalo AV, Johansson HE | chapter = Stellaris® RNA Fluorescence in Situ Hybridization for the Simultaneous Detection of Immature and Mature Long Noncoding RNAs in Adherent Cells | title = Long Non-Coding RNAs | series = Methods in Molecular Biology | volume = 1402 | pages = 119–134 | date = 2016-01-01 | pmid = 26721487 | doi = 10.1007/978-1-4939-3378-5_10 | publisher = Springer New York | isbn = 9781493933761 | veditors = Feng Y, Zhang L }}</ref> or smFISH,<ref>{{cite journal | vauthors = Chen J, McSwiggen D, Ünal E | title = Single Molecule Fluorescence In Situ Hybridization (smFISH) Analysis in Budding Yeast Vegetative Growth and Meiosis | journal = Journal of Visualized Experiments | issue = 135 | date = May 2018 | pmid = 29889208 | pmc = 6101419 | doi = 10.3791/57774 }}</ref> 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 [[hybridization probe|oligonucleotide probes]].<ref>{{cite journal | vauthors = Raj A, van den Bogaard P, Rifkin SA, van Oudenaarden A, Tyagi S | title = Imaging individual mRNA molecules using multiple singly labeled probes | journal = Nature Methods | volume = 5 | issue = 10 | pages = 877–879 | date = October 2008 | pmid = 18806792 | pmc = 3126653 | doi = 10.1038/nmeth.1253 }}</ref> The binding of up to 48 [[fluorescent label]]ed 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 noise|background]].<ref>[http://www.biosearchtech.com/display.aspx?catid=227&pageid=215 Biosearch Technologies Signs Exclusive License for Single Molecule FISH Technologies from UMDNJ]. biosearchtech.com</ref>
Single-molecule RNA FISH assays can be performed in simplex or [[Multiplex (assay)|multiplex]], and can be used as a follow-up experiment to [[quantitative PCR]], or imaged simultaneously with a [[immunohistochemistry|fluorescent antibody]] assay. The technology has potential applications in [[cancer diagnosis]],<ref>{{cite journal | vauthors = Cagir B, Gelmann A, Park J, Fava T, Tankelevitch A, Bittner EW, Weaver EJ, Palazzo JP, Weinberg D, Fry RD, Waldman SA | display-authors = 6 | title = Guanylyl cyclase C messenger RNA is a biomarker for recurrent stage II colorectal cancer | journal = Annals of Internal Medicine | volume = 131 | issue = 11 | pages = 805–812 | date = December 1999 | pmid = 10610624 | doi = 10.7326/0003-4819-131-11-199912070-00024 }}</ref> [[neuroscience]], [[gene expression]] analysis,<ref name="pmid15297669">{{cite journal | vauthors = Kosman D, Mizutani CM, Lemons D, Cox WG, McGinnis W, Bier E | title = Multiplex detection of RNA expression in Drosophila embryos | journal = Science | volume = 305 | issue = 5685 | pages = 846 | date = August 2004 | pmid = 15297669 | doi = 10.1126/science.1099247 | s2cid = 26313219 }}</ref> and [[personalized medicine|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 [[Shearing (physics)|shear]] along the length of the slide, either to cells that have been fixed to the slide and then [[Lysis|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 [[kilobase]]s. The preparation of fiber FISH samples, although conceptually simple, is a rather skilled art, and only specialized laboratories use the technique routinely.<ref>{{cite journal | vauthors = Heiskanen M, Kallioniemi O, Palotie A | title = Fiber-FISH: experiences and a refined protocol | journal = Genetic Analysis | volume = 12 | issue = 5–6 | pages = 179–184 | date = March 1996 | pmid = 8740834 | doi = 10.1016/S1050-3862(96)80004-0 }}</ref>
===Q-FISH===
{{main|Q-FISH}}
Q-FISH combines FISH with [[Peptide nucleic acid|PNAs]] and computer software to quantify fluorescence intensity. This technique is used routinely in [[telomere]] length research.
===Flow-FISH===
{{main|Flow-FISH}}
Flow-FISH uses [[flow cytometry]] to perform FISH automatically using per-cell fluorescence measurements.
===MA-FISH===
Microfluidics-assisted FISH ([http://www.nature.com/labinvest/journal/v97/n1/abs/labinvest2016121a.html 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/neu|''HER2'']] gene in breast cancer tissues.<ref>{{cite journal | vauthors = Nguyen HT, Trouillon R, Matsuoka S, Fiche M, de Leval L, Bisig B, Gijs MA | title = Microfluidics-assisted fluorescence in situ hybridization for advantageous human epidermal growth factor receptor 2 assessment in breast cancer | journal = Laboratory Investigation; A Journal of Technical Methods and Pathology | volume = 97 | issue = 1 | pages = 93–103 | date = January 2017 | pmid = 27892928 | doi = 10.1038/labinvest.2016.121 | doi-access = free }}</ref>
===MAR-FISH===
Microautoradiography FISH is a technique to combine radio-labeled substrates with conventional FISH to detect phylogenetic groups and metabolic activities simultaneously.<ref>{{cite journal| vauthors = Okabe S, Kindaichi T, Ito T |date=2004 |title=MAR-FISH—An Ecophysiological Approach to Link Phylogenetic Affiliation and In Situ Metabolic Activity of Microorganisms at a Single-Cell Resolution |journal=Microbes and Environments |volume=19 |issue=2 |pages=83–98 |doi=10.1264/jsme2.19.83 |doi-access=free}}</ref>
===Hybrid Fusion-FISH===
Hybrid Fusion FISH ([http://www.prevnos.com/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<ref>{{cite journal | vauthors = Chen KH, Boettiger AN, Moffitt JR, Wang S, Zhuang X | title = RNA imaging. Spatially resolved, highly multiplexed RNA profiling in single cells | journal = Science | volume = 348 | issue = 6233 | pages = aaa6090 | date = April 2015 | pmid = 25858977 | pmc = 4662681 | doi = 10.1126/science.aaa6090 }}</ref> is a highly multiplexed version of smFISH. It uses combinatorial labeling, followed by imaging, and then error-resistant encoding<ref>{{cite journal | vauthors = Chen KH, Boettiger AN, Moffitt JR, Wang S, Zhuang X | title = RNA imaging. Spatially resolved, highly multiplexed RNA profiling in single cells | journal = Science | volume = 348 | issue = 6233 | pages = aaa6090 | date = April 2015 | pmid = 25858977 | pmc = 4662681 | doi = 10.1126/science.aaa6090 }}</ref> to capture a high number of RNA molecules and spatial localization within the cell. The capture of a large number of RNA molecules enables elucidation of gene regulatory networks, prediction of function of unannotated genes, and identification of RNA molecule distribution patterns, which correlate with their associated proteins.
=== STARFISH ===
Starfish is a set of software tools developed in 2019 by a consortium of scientists to analyze data from nine different variations of FISH, since all variations produce the same set of data—gene expression values mapped to x and y coordinates in a cell. The software, created for all scientists, not just bioinformaticians, reads a set of images, removes noise, and identifies RNA molecules. This approach has set out to define a standard analysis scheme of FISH datasets in a similar way to [[single-cell transcriptomics]] analysis.<ref>{{cite journal | vauthors = Perkel JM | title = Starfish enterprise: finding RNA patterns in single cells | journal = Nature | volume = 572 | issue = 7770 | pages = 549–551 | date = August 2019 | pmid = 31427807 | doi = 10.1038/d41586-019-02477-9 | s2cid = 201064966 | bibcode = 2019Natur.572..549P }}</ref>
== 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 [[cytogenetics|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 cell]]s 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.<ref name=sarrate>{{cite journal | vauthors = Sarrate Z, Vidal F, Blanco J | title = Role of sperm fluorescent in situ hybridization studies in infertile patients: indications, study approach, and clinical relevance | journal = Fertility and Sterility | volume = 93 | issue = 6 | pages = 1892–1902 | date = April 2010 | pmid = 19254793 | doi = 10.1016/j.fertnstert.2008.12.139 | doi-access = free }}</ref> The analysis of chromosomes 21, X, and Y is enough to identify oligozoospermic individuals at risk.<ref name=sarrate/>
In medicine, FISH can be used to form a [[diagnosis]], to evaluate [[prognosis]], or to evaluate [[Remission (medicine)|remission]] of a disease, such as [[cancer]].  Treatment can then be specifically tailored. A traditional exam involving metaphase chromosome analysis is often unable to identify features that distinguish one disease from another, due to subtle chromosomal features; FISH can elucidate these differences. FISH can also be used to detect diseased cells more easily than standard [[Cytogenetic]] methods, which require dividing cells and requires labor and time-intensive manual preparation and analysis of the slides by a technologist.  FISH, on the other hand, does not require living cells and can be quantified automatically, a computer counts the fluorescent dots present. However, a trained technologist is required to distinguish subtle differences in banding patterns on bent and twisted metaphase chromosomes. FISH can be incorporated into [[Lab-on-a-chip]] microfluidic device. This technology is still in a developmental stage but, like other lab on a chip methods, it may lead to more portable diagnostic techniques.<ref>{{cite book | vauthors = Kurz CM, Moosdijk SV, Thielecke H, Velten T | title = 2011 Annual International Conference of the IEEE Engineering in Medicine and Biology Society | chapter = Towards a cellular multi-parameter analysis platform: Fluorescence in situ hybridization (FISH) on microhole-array chips | volume = 2011 | pages = 8408–8411 | year = 2011 | pmid = 22256298 | doi = 10.1109/IEMBS.2011.6092074 | isbn = 978-1-4577-1589-1 | s2cid = 4955677 }}</ref><ref>{{cite book | title=Microarrays: Preparation, Microfluidics, Detection Methods, and Biological Applications | publisher=Springer | veditors = Dill K, Liu R, Grodzinsky P | year=2008 | pages=323 | isbn=978-0387727165}}</ref>
[[File:FISH for Bacterial Pathogen Identification.png|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.<ref name=":0">{{cite journal | vauthors = Frickmann H, Zautner AE, Moter A, Kikhney J, Hagen RM, Stender H, Poppert S | title = Fluorescence in situ hybridization (FISH) in the microbiological diagnostic routine laboratory: a review | journal = Critical Reviews in Microbiology | volume = 43 | issue = 3 | pages = 263–293 | date = May 2017 | pmid = 28129707 | doi = 10.3109/1040841X.2016.1169990 | s2cid = 25252460 }}</ref> 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.<ref name=":0" />
FISH can also be used to compare the genomes of two biological [[species (biology)|species]], to deduce [[evolution]]ary relationships.  A similar hybridization technique is called a [[zoo blot]]. Bacterial FISH probes are often primers for the [[16s rRNA]] region.
FISH is widely used in the field of [[microbial ecology]], to identify [[microorganisms]]. [[Biofilms]], for example, are composed of complex (often) multi-species bacterial organizations. Preparing DNA probes for one species and performing FISH with this probe allows one to visualize the distribution of this specific species within the biofilm. Preparing probes (in two different colors) for two species allows researchers to visualize/study co-localization of these two species in the biofilm and can be useful in determining the fine architecture of the biofilm.
===Comparative genomic hybridization===
[[Comparative genomic hybridization]] can be described as a method that uses FISH in a parallel manner with the comparison of the hybridization strength to recall any major disruptions in the duplication process of the DNA sequences in the genome of the nucleus.<ref>{{cite web|title=Comparative Genomic Hybridization|url=http://www.credoreference.com/entry/mhscience/comparative_genomic_hybridization|publisher=McGraw-Hill Dictionary of Scientific and Technical Terms|access-date=September 19, 2013}}</ref>
=== Virtual karyotype ===
[[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 ==
[[File:Human metaphase painted with mouse chromosome 11 probe.png|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.<ref name=":1">{{cite journal | vauthors = Ferguson-Smith MA, Pereira JC, Borges A, Kasai F | title = Observations on chromosome-specific sequencing for the construction of cross-species chromosome homology maps and its resolution of human:alpaca homology | journal = Molecular Cytogenetics | volume = 15 | issue = 1 | pages = 44 | date = October 2022 | pmid = 36207754 | pmc = 9547437 | doi = 10.1186/s13039-022-00622-0 | doi-access = free }}</ref>]]
FISH can be used to study the [[evolution]] of [[Chromosome|chromosomes]]. Species that are related have similar chromosomes. This homology can be detected by [[gene]] or [[Whole genome sequencing|genome sequencing]] but also by FISH. For instance, [[human]] and [[chimpanzee]] chromosomes are very similar and FISH can demonstrate that two chimpanzee chromosomes fused to result in one human chromosome. Similarly, species that are more distantly related, have similar chromosomes but with increasing distance chromosomes tend to break and fuse and thus result in mosaic chromosomes. This can be impressively demonstrated by FISH (see figure).<ref name=":1" />
== See also ==
* [[Chromogenic in situ hybridization]] (CISH)
* [[Eukaryotic chromosome fine structure]]
* [[G banding]]
* [[Gene mapping]]
* [[Genome evolution]]
* [[Happy mapping]]
* [[In situ hybridization]], the technique used for labelling
* [[Molecular cytogenetics]]
* [[Virtual karyotype]]
== Gallery ==
<gallery>
Image:FISH (technique).gif|Another schematic of FISH process.
Image:FISHchip.jpg|Microfluidic chip that lowered the cost-per-test of FISH by 90%.
Image:BacteriaFISH.jpg|Dual label FISH image; Bifidobacteria Cy3, Total bacteria FITC.
Image:NEAT1 paraspeckles in U-2 OS cells.jpg|Paraspeckles visualized by single-molecule FISH against NEAT1 (Quasar 570) in U-2 OS cells (DAPI).
</gallery>
== References ==
{{Reflist|30em}}
== Further reading ==
{{refbegin}}
* {{cite journal | vauthors = Pernthaler A, Pernthaler J, Amann R | title = Fluorescence in situ hybridization and catalyzed reporter deposition for the identification of marine bacteria | journal = Applied and Environmental Microbiology | volume = 68 | issue = 6 | pages = 3094–3101 | date = June 2002 | pmid = 12039771 | pmc = 123953 | doi = 10.1128/AEM.68.6.3094-3101.2002 | bibcode = 2002ApEnM..68.3094P }}
* {{cite journal | vauthors = Wagner M, Horn M, Daims H | title = Fluorescence in situ hybridisation for the identification and characterisation of prokaryotes | journal = Current Opinion in Microbiology | volume = 6 | issue = 3 | pages = 302–309 | date = June 2003 | pmid = 12831908 | doi = 10.1016/S1369-5274(03)00054-7 }}
* {{cite book| vauthors = Carthy JD |title=Viewpoints In Biology|year=1965|publisher=Butterworth & Co.|location=England|page=66}}
{{refend}}
== External links ==
{{Commons category}}
* {{MeshName|Fluorescent+in+Situ+Hybridization}}
* Information on [https://web.archive.org/web/20060621160133/http://olympusfluoview.com/applications/fiberfish.html fiber FISH] from the [[Olympus Corporation]]
* A [http://info.med.yale.edu/genetics/ward/tavi/fi19.html guide to fiber FISH] from Octavian Henegariu
* [http://www.sanger.ac.uk/HGP/methods/cytogenetics/fiber_fish.shtml Fibre FISH protocol] {{Webarchive|url=https://web.archive.org/web/20061023232719/http://www.sanger.ac.uk/HGP/methods/cytogenetics/fiber_fish.shtml |date=2006-10-23 }} from the [[Human Genome Project]] at the [[Sanger Centre]]
* [http://wiki.biomine.skelleftea.se/wiki/index.php/CARD-FISH CARD-FISH, BioMineWiki] {{Webarchive|url=https://web.archive.org/web/20200728114942/http://wiki.biomine.skelleftea.se/wiki/index.php/CARD-FISH |date=2020-07-28 }}
* [http://www.cshprotocols.org/cgi/content/full/2007/10/pdb.prot4730 Preparation of Complex DNA Probe Sets for 3D FISH with up to Six Different Fluorochromes]
* [http://www.genedetect.com/insitu.htm FISH technical notes and protocols from GeneDetect.com]
* [http://www.biovisible.com/photopage.shtml Fluorescence in situ Hybridization Photos of bacteria] {{Webarchive|url=https://web.archive.org/web/20150205200138/http://www.biovisible.com/photopage.shtml |date=2015-02-05 }}
* Rational design of polynucleotide probe mixes to identify particular genes in defined taxa: www.dnaBaser.com/PolyPro
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[[Category:Anatomical pathology]]
[[Category:Cytogenetics]]
[[Category:Laboratory techniques]]
[[Category:Molecular biology]]
[[Category:Gene tests]]
[[Category:Pathology]]
[[Category:Nuclear organization]]