In Situ Hybridization (ISH) is a technique that allows for precise localization of a specific segment of nucleic acid within a histologic section. The underlying basis of ISH is that nucleic acids, if preserved adequately within a histologic specimen, can be detected through the application of a complementary strand of nucleic acid to which a reporter molecule is attached.
Visualization of the reporter molecule allows localizing DNA or RNA sequences in a heterogeneous cell populations including tissue samples and environmental samples. Riboprobes also allow to localize and assess degree of gene expression. The technique is particularly useful in neuroscience.
In situ hybridization (ISH) is a type of hybridization that uses a labeled complementary DNA or RNA strand (i.e., probe) to localize a specific DNA or RNA sequence in a portion or section of tissue (in situ), or, if the tissue is small enough (e.g. plant seeds, Drosophila embryos), in the entire tissue (whole mount ISH). This is distinct from immunohistochemistry, which usually localizes proteins in tissue sections. DNA ISH can be used to determine the structure of chromosomes. Fluorescent DNA ISH (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.
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 DNA or, now most commonly, a complementary RNA (riboprobe). 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) is localized and quantified in the tissue using either autoradiography, fluorescence microscopy or immunohistochemistry, 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.
In situ hybridization probes
• double-stranded DNA (dsDNA) probes
• single-stranded DNA (ssDNA) probes
• RNA probes (riboprobes)
• synthetic oligonucleotides (PNA, LNA)
• radioactive isotopes
• non-radioactive labels
o fluorescent dye (FISH)
Applications of In Situ Hybridization• microbiology (classic target - 16S rRNA) - morphology and population structure of microorganisms
• pathology (pathogen profiling, abnormal gene expression)
• developmental biology (gene expression profiling in embryonic tissues)
• karyotyping and phylogenetic analysis (unique FISH patterns on individual chromosomes, chromosomal abberations)
• physical mapping (mapping clones on chromosomes and direct assignment of mapped clones to chromosomal regions associated with heterochromatin or euchromatin)
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