Fluorescence in situ hybridization

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Multiplex RNA visualization in cells using ViewRNA FISH Assays Multiplex ViewRNA FISH Assay in Jurkat and HeLa cells.jpg
Multiplex RNA visualization in cells using ViewRNA FISH Assays
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. Bcrablmet.jpg
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 only particular parts of a nucleic acid sequence with a high degree of sequence complementarity. It was developed by biomedical researchers in the early 1980s [1] to detect and localize the presence or absence of specific DNA sequences on chromosomes. 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. [2] FISH can also be used to detect and localize specific RNA targets (mRNA, lncRNA and miRNA)[ citation needed ] 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.

Contents

Probes – RNA and DNA

ViewRNA detection of miR-133(green) and myogenin mRNA (red) in C2C12 differentiating cells MicroRNA and mRNA visualization in differentiating C1C12 cells.jpg
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, [3] [4] [5] lncRNA [6] [7] [8] and miRNA in tissues and cells. FISH is used by examining the cellular reproduction cycle, specifically interphase of the nuclei for any chromosomal abnormalities. [9] 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. [9] The hybridization signals for each probe when a nucleic abnormality is detected. [9] 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.

Urothelial cells marked with four different probes Urovysion on Duet.png
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, 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 (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. [10] 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. Some commonly used fixatives are 4% formaldehyde or paraformaldehyde (PFA) in phosphate buffered saline (PBS). [10] FISH has also been successfully done on unfixed cells. [11] 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. [12]

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. [13]

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. [14] 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. [12]

Preparation and hybridization process – DNA

Scheme of the principle of the FISH Experiment to localize a gene in the nucleus. FISH (Fluorescent In Situ Hybridization).jpg
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 tagged directly with fluorophores, with targets for antibodies or with biotin. Tagging can be done in various ways, such as nick translation, or polymerase chain reaction using tagged nucleotides.

Then, an interphase or metaphase chromosome preparation is produced. The chromosomes are firmly attached to a 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 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 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, 14, 21, 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 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 [15] or smFISH, [16] 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. [17] 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. [18]

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, [19] neuroscience, gene expression analysis, [20] 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. [21]

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. [22]

MAR-FISH

Microautoradiography FISH is a technique to combine radio-labeled substrates with conventional FISH to detect phylogenetic groups and metabolic activities simultaneously. [23]

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 [24] is a highly multiplexed version of smFISH. It uses combinatorial labeling, followed by imaging, and then error-resistant encoding [25] 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. [26]

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. [27] The analysis of chromosomes 21, X, and Y is enough to identify oligozoospermic individuals at risk. [27]

In medicine, FISH can be used to form a diagnosis, to evaluate prognosis, or to evaluate 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. [28] [29]

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. FISH for Bacterial Pathogen Identification.png
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.

Species identification

FISH has been extensively studied as a diagnostic technique for the identification of pathogens in the field of medical microbiology. [30] 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. [30]

FISH can also be used to compare the genomes of two biological species, to deduce evolutionary 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. [31]

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

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. Human metaphase painted with mouse chromosome 11 probe.png
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 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). [32]

See also

Related Research Articles

<span class="mw-page-title-main">Southern blot</span> DNA analysis technique

Southern blot is a method used for detection and quantification of a specific DNA sequence in DNA samples. This method is used in molecular biology. Briefly, purified DNA from a biological sample is digested with restriction enzymes, and the resulting DNA fragments are separated by electrophoresis using an electric current to move them through a sieve-like gel or matrix, which allows smaller fragments to move faster than larger fragments. The DNA fragments are transferred out of the gel or matrix onto a solid membrane, which is then exposed to a DNA probe labeled with a radioactive, fluorescent, or chemical tag. The tag allows any DNA fragments containing complementary sequences with the DNA probe sequence to be visualized within the Southern blot.

<span class="mw-page-title-main">DNA microarray</span> Collection of microscopic DNA spots attached to a solid surface

A DNA microarray is a collection of microscopic DNA spots attached to a solid surface. Scientists use DNA microarrays to measure the expression levels of large numbers of genes simultaneously or to genotype multiple regions of a genome. Each DNA spot contains picomoles of a specific DNA sequence, known as probes. These can be a short section of a gene or other DNA element that are used to hybridize a cDNA or cRNA sample under high-stringency conditions. Probe-target hybridization is usually detected and quantified by detection of fluorophore-, silver-, or chemiluminescence-labeled targets to determine relative abundance of nucleic acid sequences in the target. The original nucleic acid arrays were macro arrays approximately 9 cm × 12 cm and the first computerized image based analysis was published in 1981. It was invented by Patrick O. Brown. An example of its application is in SNPs arrays for polymorphisms in cardiovascular diseases, cancer, pathogens and GWAS analysis. It is also used for the identification of structural variations and the measurement of gene expression.

<span class="mw-page-title-main">Fluorescent tag</span>

In molecular biology and biotechnology, a fluorescent tag, also known as a fluorescent label or fluorescent probe, is a molecule that is attached chemically to aid in the detection of a biomolecule such as a protein, antibody, or amino acid. Generally, fluorescent tagging, or labeling, uses a reactive derivative of a fluorescent molecule known as a fluorophore. The fluorophore selectively binds to a specific region or functional group on the target molecule and can be attached chemically or biologically. Various labeling techniques such as enzymatic labeling, protein labeling, and genetic labeling are widely utilized. Ethidium bromide, fluorescein and green fluorescent protein are common tags. The most commonly labelled molecules are antibodies, proteins, amino acids and peptides which are then used as specific probes for detection of a particular target.

Comparative genomic hybridization (CGH) is a molecular cytogenetic method for analysing copy number variations (CNVs) relative to ploidy level in the DNA of a test sample compared to a reference sample, without the need for culturing cells. The aim of this technique is to quickly and efficiently compare two genomic DNA samples arising from two sources, which are most often closely related, because it is suspected that they contain differences in terms of either gains or losses of either whole chromosomes or subchromosomal regions. This technique was originally developed for the evaluation of the differences between the chromosomal complements of solid tumor and normal tissue, and has an improved resolution of 5–10 megabases compared to the more traditional cytogenetic analysis techniques of giemsa banding and fluorescence in situ hybridization (FISH) which are limited by the resolution of the microscope utilized.

In molecular biology, a hybridization probe (HP) is a fragment of DNA or RNA, usually 15–10000 nucleotides long, which can be radioactively or fluorescently labeled. HPs can be used to detect the presence of nucleotide sequences in analyzed RNA or DNA that are complementary to the sequence in the probe. The labeled probe is first denatured into single stranded DNA (ssDNA) and then hybridized to the target ssDNA or RNA immobilized on a membrane or in situ.

<span class="mw-page-title-main">Fusion gene</span> Gene formed by the combination of two independent genes

In genetics, a fusion gene is a hybrid gene formed from two previously independent genes. It can occur as a result of translocation, interstitial deletion, or chromosomal inversion. Fusion genes have been found to be prevalent in all main types of human neoplasia. The identification of these fusion genes play a prominent role in being a diagnostic and prognostic marker.

<i>In situ</i> hybridization Laboratory technique to localize nucleic acids

In situ hybridization (ISH) is a type of hybridization that uses a labeled complementary DNA, RNA or modified nucleic acid strand to localize a specific DNA or RNA sequence in a portion or section of tissue or if the tissue is small enough, in the entire tissue, in cells, and in circulating tumor cells (CTCs). This is distinct from immunohistochemistry, which usually localizes proteins in tissue sections.

<span class="mw-page-title-main">Gene mapping</span> Process of locating specific genes

Gene mapping or genome mapping describes the methods used to identify the location of a gene on a chromosome and the distances between genes. Gene mapping can also describe the distances between different sites within a gene.

<span class="mw-page-title-main">Real-time polymerase chain reaction</span> Laboratory technique of molecular biology

A real-time polymerase chain reaction is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR). It monitors the amplification of a targeted DNA molecule during the PCR, not at its end, as in conventional PCR. Real-time PCR can be used quantitatively and semi-quantitatively.

A dicentric chromosome is an abnormal chromosome with two centromeres. It is formed through the fusion of two chromosome segments, each with a centromere, resulting in the loss of acentric fragments and the formation of dicentric fragments. The formation of dicentric chromosomes has been attributed to genetic processes, such as Robertsonian translocation and paracentric inversion. Dicentric chromosomes have important roles in the mitotic stability of chromosomes and the formation of pseudodicentric chromosomes. Their existence has been linked to certain natural phenomena such as irradiation and have been documented to underlie certain clinical syndromes, notably Kabuki syndrome. The formation of dicentric chromosomes and their implications on centromere function are studied in certain clinical cytogenetics laboratories.

<span class="mw-page-title-main">Molecular cytogenetics</span>

Molecular cytogenetics combines two disciplines, molecular biology and cytogenetics, and involves the analysis of chromosome structure to help distinguish normal and cancer-causing cells. Human cytogenetics began in 1956 when it was discovered that normal human cells contain 46 chromosomes. However, the first microscopic observations of chromosomes were reported by Arnold, Flemming, and Hansemann in the late 1800s. Their work was ignored for decades until the actual chromosome number in humans was discovered as 46. In 1879, Arnold examined sarcoma and carcinoma cells having very large nuclei. Today, the study of molecular cytogenetics can be useful in diagnosing and treating various malignancies such as hematological malignancies, brain tumors, and other precursors of cancer. The field is overall focused on studying the evolution of chromosomes, more specifically the number, structure, function, and origin of chromosome abnormalities. It includes a series of techniques referred to as fluorescence in situ hybridization, or FISH, in which DNA probes are labeled with different colored fluorescent tags to visualize one or more specific regions of the genome. Introduced in the 1980s, FISH uses probes with complementary base sequences to locate the presence or absence of the specific DNA regions. FISH can either be performed as a direct approach to metaphase chromosomes or interphase nuclei. Alternatively, an indirect approach can be taken in which the entire genome can be assessed for copy number changes using virtual karyotyping. Virtual karyotypes are generated from arrays made of thousands to millions of probes, and computational tools are used to recreate the genome in silico.

Chromosome combing is a technique used to produce an array of uniformly stretched DNA that is then highly suitable for nucleic acid hybridization studies such as fluorescent in situ hybridisation (FISH) which benefit from the uniformity of stretching, the easy access to the hybridisation target sequences, and the resolution offered by the large distance between two probes, which is due to the stretching of the DNA by a factor of 1.5 times the crystallographic length of DNA.

Flow-FISH is a cytogenetic technique to quantify the copy number of RNA or specific repetitive elements in genomic DNA of whole cell populations via the combination of flow cytometry with cytogenetic fluorescent in situ hybridization staining protocols.

<span class="mw-page-title-main">Tiling array</span>

Tiling arrays are a subtype of microarray chips. Like traditional microarrays, they function by hybridizing labeled DNA or RNA target molecules to probes fixed onto a solid surface.

A Riboprobe, abbreviation of RNA probe, is a segment of labelled RNA that can be used to detect a target mRNA or DNA during in situ hybridization. RNA probes can be produced by in vitro transcription of cloned DNA inserted in a suitable plasmid downstream of a viral promoter. Some bacterial viruses code for their own RNA polymerases, which are highly specific for the viral promoters. Using these enzymes, labeled NTPs, and inserts inserted in both forward and reverse orientations, both sense and antisense riboprobes can be generated from a cloned gene.

Chromogenic in situ hybridization (CISH) is a cytogenetic technique that combines the chromogenic signal detection method of immunohistochemistry (IHC) techniques with in situ hybridization. It was developed around the year 2000 as an alternative to fluorescence in situ hybridization (FISH) for detection of HER-2/neu oncogene amplification. CISH is similar to FISH in that they are both in situ hybridization techniques used to detect the presence or absence of specific regions of DNA. However, CISH is much more practical in diagnostic laboratories because it uses bright-field microscopes rather than the more expensive and complicated fluorescence microscopes used in FISH.

<span class="mw-page-title-main">Neuronal lineage marker</span> Endogenous tag expressed in different cells along neurogenesis and differentiated cells

A neuronal lineage marker is an endogenous tag that is expressed in different cells along neurogenesis and differentiated cells such as neurons. It allows detection and identification of cells by using different techniques. A neuronal lineage marker can be either DNA, mRNA or RNA expressed in a cell of interest. It can also be a protein tag, as a partial protein, a protein or an epitope that discriminates between different cell types or different states of a common cell. An ideal marker is specific to a given cell type in normal conditions and/or during injury. Cell markers are very valuable tools for examining the function of cells in normal conditions as well as during disease. The discovery of various proteins specific to certain cells led to the production of cell-type-specific antibodies that have been used to identify cells.

In molecular biology, hybridization is a phenomenon in which single-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules anneal to complementary DNA or RNA. Though a double-stranded DNA sequence is generally stable under physiological conditions, changing these conditions in the laboratory will cause the molecules to separate into single strands. These strands are complementary to each other but may also be complementary to other sequences present in their surroundings. Lowering the surrounding temperature allows the single-stranded molecules to anneal or “hybridize” to each other.

Spatial transcriptomics is a method for assigning cell types to their locations in the histological sections. It comprises an important part of spatial biology. Recent work demonstrated that the subcellular localization of mRNA molecules, for example, in the nucleus can also be studied.

Physical map is a technique used in molecular biology to find the order and physical distance between DNA base pairs by DNA markers. It is one of the gene mapping techniques which can determine the sequence of DNA base pairs with high accuracy. Genetic mapping, another approach of gene mapping, can provide markers needed for the physical mapping. However, as the former deduces the relative gene position by recombination frequencies, it is less accurate than the latter.

References

  1. ↑ Langer-Safer PR, Levine M, Ward DC (July 1982). "Immunological method for mapping genes on drosophila polytene chromosomes". Proceedings of the National Academy of Sciences of the United States of America. 79 (14): 4381–4385. Bibcode:1982PNAS...79.4381L. doi: 10.1073/pnas.79.14.4381 . PMC   346675 . PMID   6812046.
  2. ↑ Amann R, Fuchs BM (May 2008). "Single-cell identification in microbial communities by improved fluorescence in situ hybridization techniques". Nature Reviews. Microbiology. 6 (5): 339–348. doi:10.1038/nrmicro1888. PMID   18414500. S2CID   22498325.
  3. ↑ Anthony SJ, St Leger JA, Pugliares K, Ip HS, Chan JM, Carpenter ZW, et al. (2012). "Emergence of fatal avian influenza in New England harbor seals". mBio. 3 (4): e00166–e00112. doi:10.1128/mBio.00166-12. PMC   3419516 . PMID   22851656.
  4. ↑ Everitt AR, Clare S, Pertel T, John SP, Wash RS, Smith SE, et al. (March 2012). "IFITM3 restricts the morbidity and mortality associated with influenza". Nature. 484 (7395): 519–523. Bibcode:2012Natur.484..519.. doi:10.1038/nature10921. PMC   3648786 . PMID   22446628.
  5. ↑ Louzada S, Adega F, Chaves R (2012). "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". PLOS ONE. 7 (1): e29923. Bibcode:2012PLoSO...729923L. doi: 10.1371/journal.pone.0029923 . PMC   3254647 . PMID   22253826.
  6. ↑ Ting DT, Lipson D, Paul S, Brannigan BW, Akhavanfard S, Coffman EJ, et al. (February 2011). "Aberrant overexpression of satellite repeats in pancreatic and other epithelial cancers". Science. 331 (6017): 593–596. Bibcode:2011Sci...331..593T. doi:10.1126/science.1200801. PMC   3701432 . PMID   21233348.
  7. ↑ Zhang B, Arun G, Mao YS, Lazar Z, Hung G, Bhattacharjee G, et al. (July 2012). "The lncRNA Malat1 is dispensable for mouse development but its transcription plays a cis-regulatory role in the adult". Cell Reports. 2 (1): 111–123. doi:10.1016/j.celrep.2012.06.003. PMC   3408587 . PMID   22840402.
  8. ↑ Lee K, Kunkeaw N, Jeon SH, Lee I, Johnson BH, Kang GY, et al. (June 2011). "Precursor miR-886, a novel noncoding RNA repressed in cancer, associates with PKR and modulates its activity". RNA. 17 (6): 1076–1089. doi:10.1261/rna.2701111. PMC   3096040 . PMID   21518807.
  9. 1 2 3 Bernasconi B, Karamitopoulou-Diamantis E, Karamitopolou-Diamantiis E, Tornillo L, Lugli A, Di Vizio D, et al. (April 2008). "Chromosomal instability in gastric mucosa-associated lymphoid tissue lymphomas: a fluorescent in situ hybridization study using a tissue microarray approach". Human Pathology. 39 (4): 536–542. doi:10.1016/j.humpath.2007.08.009. PMID   18234275.
  10. 1 2 Young AP, Jackson DJ, Wyeth RC (2020-03-19). "A technical review and guide to RNA fluorescence in situ hybridization". PeerJ. 8: e8806. doi: 10.7717/peerj.8806 . PMC   7085896 . PMID   32219032.
  11. ↑ Haroon MF, Skennerton CT, Steen JA, Lachner N, Hugenholtz P, Tyson GW (2013). "In-solution fluorescence in situ hybridization and fluorescence-activated cell sorting for single cell and population genome recovery". Microbial Metagenomics, Metatranscriptomics, and Metaproteomics. Methods in Enzymology. Vol. 531. pp. 3–19. doi:10.1016/B978-0-12-407863-5.00001-0. ISBN   9780124078635. PMID   24060113.
  12. 1 2 Cui C, Shu W, Li P (2016). "Fluorescence In situ Hybridization: Cell-Based Genetic Diagnostic and Research Applications". Frontiers in Cell and Developmental Biology. 4: 89. doi: 10.3389/fcell.2016.00089 . PMC   5011256 . PMID   27656642.
  13. ↑ Xie F, Timme KA, Wood JR (May 2018). "Using Single Molecule mRNA Fluorescent in Situ Hybridization (RNA-FISH) to Quantify mRNAs in Individual Murine Oocytes and Embryos". Scientific Reports. 8 (1): 7930. Bibcode:2018NatSR...8.7930X. doi:10.1038/s41598-018-26345-0. PMC   5962540 . PMID   29785002.
  14. ↑ Oliveira VC, Carrara RC, Simoes DL, Saggioro FP, Carlotti CG, Covas DT, Neder L (August 2010). "Sudan Black B treatment reduces autofluorescence and improves resolution of in situ hybridization specific fluorescent signals of brain sections". Histology and Histopathology. 25 (8): 1017–1024. doi:10.14670/HH-25.1017. PMID   20552552.
  15. ↑ Orjalo AV, Johansson HE (2016-01-01). "Stellaris® RNA Fluorescence in Situ Hybridization for the Simultaneous Detection of Immature and Mature Long Noncoding RNAs in Adherent Cells". In Feng Y, Zhang L (eds.). Long Non-Coding RNAs. Methods in Molecular Biology. Vol. 1402. Springer New York. pp. 119–134. doi:10.1007/978-1-4939-3378-5_10 (inactive 2024-10-31). ISBN   9781493933761. PMID   26721487.{{cite book}}: CS1 maint: DOI inactive as of October 2024 (link)
  16. ↑ Chen J, McSwiggen D, Ünal E (May 2018). "Single Molecule Fluorescence In Situ Hybridization (smFISH) Analysis in Budding Yeast Vegetative Growth and Meiosis". Journal of Visualized Experiments (135). doi:10.3791/57774. PMC   6101419 . PMID   29889208.
  17. ↑ Raj A, van den Bogaard P, Rifkin SA, van Oudenaarden A, Tyagi S (October 2008). "Imaging individual mRNA molecules using multiple singly labeled probes". Nature Methods. 5 (10): 877–879. doi:10.1038/nmeth.1253. PMC   3126653 . PMID   18806792.
  18. ↑ Biosearch Technologies Signs Exclusive License for Single Molecule FISH Technologies from UMDNJ. biosearchtech.com
  19. ↑ Cagir B, Gelmann A, Park J, Fava T, Tankelevitch A, Bittner EW, et al. (December 1999). "Guanylyl cyclase C messenger RNA is a biomarker for recurrent stage II colorectal cancer". Annals of Internal Medicine. 131 (11): 805–812. doi:10.7326/0003-4819-131-11-199912070-00024. PMID   10610624.
  20. ↑ Kosman D, Mizutani CM, Lemons D, Cox WG, McGinnis W, Bier E (August 2004). "Multiplex detection of RNA expression in Drosophila embryos". Science. 305 (5685): 846. doi:10.1126/science.1099247. PMID   15297669. S2CID   26313219.
  21. ↑ Heiskanen M, Kallioniemi O, Palotie A (March 1996). "Fiber-FISH: experiences and a refined protocol". Genetic Analysis. 12 (5–6): 179–184. doi:10.1016/S1050-3862(96)80004-0. PMID   8740834.
  22. ↑ Nguyen HT, Trouillon R, Matsuoka S, Fiche M, de Leval L, Bisig B, Gijs MA (January 2017). "Microfluidics-assisted fluorescence in situ hybridization for advantageous human epidermal growth factor receptor 2 assessment in breast cancer". Laboratory Investigation; A Journal of Technical Methods and Pathology. 97 (1): 93–103. doi: 10.1038/labinvest.2016.121 . PMID   27892928.
  23. ↑ Okabe S, Kindaichi T, Ito T (2004). "MAR-FISH—An Ecophysiological Approach to Link Phylogenetic Affiliation and In Situ Metabolic Activity of Microorganisms at a Single-Cell Resolution". Microbes and Environments. 19 (2): 83–98. doi: 10.1264/jsme2.19.83 .
  24. ↑ Chen KH, Boettiger AN, Moffitt JR, Wang S, Zhuang X (April 2015). "RNA imaging. Spatially resolved, highly multiplexed RNA profiling in single cells". Science. 348 (6233): aaa6090. doi:10.1126/science.aaa6090. PMC   4662681 . PMID   25858977.
  25. ↑ Chen KH, Boettiger AN, Moffitt JR, Wang S, Zhuang X (April 2015). "RNA imaging. Spatially resolved, highly multiplexed RNA profiling in single cells". Science. 348 (6233): aaa6090. doi:10.1126/science.aaa6090. PMC   4662681 . PMID   25858977.
  26. ↑ Perkel JM (August 2019). "Starfish enterprise: finding RNA patterns in single cells". Nature. 572 (7770): 549–551. Bibcode:2019Natur.572..549P. doi:10.1038/d41586-019-02477-9. PMID   31427807. S2CID   201064966.
  27. 1 2 Sarrate Z, Vidal F, Blanco J (April 2010). "Role of sperm fluorescent in situ hybridization studies in infertile patients: indications, study approach, and clinical relevance". Fertility and Sterility. 93 (6): 1892–1902. doi: 10.1016/j.fertnstert.2008.12.139 . PMID   19254793.
  28. ↑ Kurz CM, Moosdijk SV, Thielecke H, Velten T (2011). "Towards a cellular multi-parameter analysis platform: Fluorescence in situ hybridization (FISH) on microhole-array chips". 2011 Annual International Conference of the IEEE Engineering in Medicine and Biology Society. Vol. 2011. pp. 8408–8411. doi:10.1109/IEMBS.2011.6092074. ISBN   978-1-4577-1589-1. PMID   22256298. S2CID   4955677.
  29. ↑ Dill K, Liu R, Grodzinsky P, eds. (2008). Microarrays: Preparation, Microfluidics, Detection Methods, and Biological Applications. Springer. p. 323. ISBN   978-0387727165.
  30. 1 2 Frickmann H, Zautner AE, Moter A, Kikhney J, Hagen RM, Stender H, Poppert S (May 2017). "Fluorescence in situ hybridization (FISH) in the microbiological diagnostic routine laboratory: a review". Critical Reviews in Microbiology. 43 (3): 263–293. doi:10.3109/1040841X.2016.1169990. PMID   28129707. S2CID   25252460.
  31. ↑ "Comparative Genomic Hybridization". McGraw-Hill Dictionary of Scientific and Technical Terms. Retrieved September 19, 2013.
  32. 1 2 Ferguson-Smith MA, Pereira JC, Borges A, Kasai F (October 2022). "Observations on chromosome-specific sequencing for the construction of cross-species chromosome homology maps and its resolution of human:alpaca homology". Molecular Cytogenetics. 15 (1): 44. doi: 10.1186/s13039-022-00622-0 . PMC   9547437 . PMID   36207754.

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