In situ hybridization
Encyclopedia
Complementary DNA
In genetics, complementary DNA is DNA synthesized from a messenger RNA template in a reaction catalyzed by the enzyme reverse transcriptase and the enzyme DNA polymerase. cDNA is often used to clone eukaryotic genes in prokaryotes...
or RNA
RNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
strand (i.e., probe
Hybridization probe
In molecular biology, a hybridization probe is a fragment of DNA or RNA of variable length , which is used in DNA or RNA samples to detect the presence of nucleotide sequences that are complementary to the sequence in the probe...
) to localize a specific DNA or RNA sequence in a portion or section of tissue
Tissue (biology)
Tissue is a cellular organizational level intermediate between cells and a complete organism. A tissue is an ensemble of cells, not necessarily identical, but from the same origin, that together carry out a specific function. These are called tissues because of their identical functioning...
(in situ
In situ
In situ is a Latin phrase which translated literally as 'In position'. It is used in many different contexts.-Aerospace:In the aerospace industry, equipment on board aircraft must be tested in situ, or in place, to confirm everything functions properly as a system. Individually, each piece may...
), or, if the tissue is small enough (e.g. plant seeds, Drosophila
Drosophila
Drosophila is a genus of small flies, belonging to the family Drosophilidae, whose members are often called "fruit flies" or more appropriately pomace flies, vinegar flies, or wine flies, a reference to the characteristic of many species to linger around overripe or rotting fruit...
embryos), in the entire tissue (whole mount ISH). This is distinct from immunohistochemistry
Immunohistochemistry
Immunohistochemistry or IHC refers to the process of detecting antigens in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues. IHC takes its name from the roots "immuno," in reference to antibodies used in the procedure, and...
, which usually localizes proteins in tissue sections. DNA ISH can be used to determine the structure
Gene mapping
Gene mapping, also called genome mapping, is the creation of a genetic map assigning DNA fragments to chromosomes.When a genome is first investigated, this map is nonexistent. The map improves with the scientific progress and is perfect when the genomic DNA sequencing of the species has been...
of chromosomes. Fluorescent DNA ISH
Fluorescent in situ hybridization
FISH is a cytogenetic technique developed by biomedical researchers in the early 1980s that is used to detect and localize the presence or absence of specific DNA sequences on chromosomes. FISH uses fluorescent probes that bind to only those parts of the chromosome with which they show a high...
(FISH) can, for example, be used in medical diagnostics to assess chromosomal integrity. RNA ISH (hybridization histochemistry) is used to measure and localize mRNAs and other transcripts within tissue sections or whole mounts.
Process
For hybridization histochemistry, sample cells and tissues are usually treated to fix the target transcripts in place and to increase access of the probe. As noted above, the probe is either a labeled complementary DNAComplementary DNA
In genetics, complementary DNA is DNA synthesized from a messenger RNA template in a reaction catalyzed by the enzyme reverse transcriptase and the enzyme DNA polymerase. cDNA is often used to clone eukaryotic genes in prokaryotes...
or, now most commonly, a complementary RNA
RNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
(riboprobe
Riboprobe
Riboprobes are RNA probes that 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...
). The probe hybridizes to the target sequence at elevated temperature, and then the excess probe is washed away (after prior hydrolysis using RNase in the case of unhybridized, excess RNA probe). Solution parameters such as temperature, salt and/or detergent concentration can be manipulated to remove any non-identical interactions (i.e. only exact sequence matches will remain bound). Then, the probe that was labeled with either radio-, fluorescent- or antigen-labeled bases (e.g., digoxigenin
Digoxigenin
Digoxigenin is a steroid found exclusively in the flowers and leaves of the plants Digitalis purpurea, Digitalis orientalis and Digitalis lanata , where it is attached to sugars, to form the glycosides Digoxigenin (DIG) is a steroid found exclusively in the flowers and leaves of the plants...
) is localized and quantified in the tissue using either autoradiography, fluorescence microscopy or immunohistochemistry
Immunohistochemistry
Immunohistochemistry or IHC refers to the process of detecting antigens in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues. IHC takes its name from the roots "immuno," in reference to antibodies used in the procedure, and...
, respectively. ISH can also use two or more probes, labeled with radioactivity or the other non-radioactive labels, to simultaneously detect two or more transcripts.
Basic Steps for Digoxigenin-labeled probes
- permeabilisation of cells with proteinase KProteinase KProteinase K is a broad-spectrum serine protease. The enzyme was discovered in 1974 in extracts of the fungus Engyodontium album . Proteinase K is able to digest native keratin , hence, the name "Proteinase K"...
to open cell membranes (around 25 minutes, not needed for tissue sections or some early-stage embryos) - binding of mRNAs to marked RNA probe (usually overnight)
- antibody-phosphatase binding to RNA-probe (some hours)
- staining of antibody (e.g. with alkaline phosphatase)
The protocol takes around 2-3 days and takes some time to set up. Some companies sell robots to automate the process. As a result, large-scale screenings have been conducted in laboratories on thousands of genes. The results can usually be accessed via websites (see external links).
External links
- Whole-Mount In Situ Hybridization of RNA Probes to Plant Tissues
- Preparation of Complex DNA Probe Sets for 3D FISH with up to Six Different Fluorochromes
- Transcript In Situ Hybridization of Whole-Mount Embryos for Phenotype Analysis of RNAi-Treated Drosophila