User:Mk21bf21/Fluorescence in situ hybridization

 Single-molecule RNA FISH[edit] 

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 longer probes hybridize less specifically than shorter probes, so that short 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[edit]
Single-molecule RNA FISH, also known as Stellaris® RNA FISH 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.

Fiber FISH[edit]
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[edit]
Main article: 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[edit]
Main article: Flow-FISH

Flow-FISH uses flow cytometry to perform FISH automatically using per-cell fluorescence measurements.

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

Hybrid Fusion-FISH[edit]
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 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 an analysis scheme of FISH datasets in a similar way to single-cell transcriptomics analysis.