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In Situ Hybridization
What is In Situ Hybridization?
In Situ Hybridization (ISH) uses a labeled probe to detect and localize specific RNA or DNA sequences in a tissue or on a chromosome. ISH relies on DNA's ability to reanneal, or hybridize, with a complimentary strand when at the correct temperature. “In situ” means “in the original place” in Latin, so ISH involves a labeled nucleic acid probe hybridizing with a DNA or RNA sequence in situ (in the cells) so that the location of the sequence of interest can be detected in the cell, tissue, or chromosome. (Immuno-ISH, http://home.no.net/immuno/). Like Northern and Southern Blots, ISH indicates the presence of a particular RNA or DNA sequence, but ISH differs from blots in that the labeled probe reveals the actual location of the sequence in the cells. ISH is the only procedure that allows the location of the sequence of interest to be studied (Polak et al., 1990). The probe can be either radioactively labeled and detected by autoradiography or fluorescently labeled (abbreviated FISH) and detected by immunocytochemistry. The specificity of the probe depends on the permeability of the cells, the type of probe, the labeling technique, and the hybridization conditions, so specificity of ISH can be adjusting according to the desired results (Polak et al. , 1990).
How does ISH work?
The general procedure for ISH involves fixing samples of chromosomes or tissues onto a glass slide. The material is then treated with chemicals to permeabilize the cells or tissues and denature the DNA so that the probe can more readily hybridize. A complementary nucleic acid probe is prepared and labeled, either by radioactivity or by fluorescence. A hybridization solution containing the probe is sloshed over the slide so that the probe can hybridize with the sequence of interest. The excess probe is washed away and then either autoradiography or immunocytochemistry is used to detect the location of the probe. Microscopy can be used to view the samples (Leitch et al., 1994). See Figure 1 for a flow chart of FISH.
Figure 1. Fluorescent In Situ Hybridization Flow Chart. The samples are fixed onto coverslips, fluorescently labeled oligonucleotide probes are hybridized with the sample, excess probes are wahed away, and epifluorescence microscopy is used to view the location of the hybrized cells. (Permission from Dr. Frank Oliver Glockner for use of image found at http://www.mpi-bremen.de/molecol/fog/fish1.html.)
Fixation and Denaturation of Material
The DNA or RNA must be fixed onto a glass slide for stability. so as to allow the probe sufficient access to the targe maintain the structure as much as possible while giving the probe sufficient access to the target by permeabilizing the cells. While DNA is relatively stable and can be fixed easily, RNA is highly degradable, so extra care must be taken to fix the mRNA onto the slide as soon as possible after it is extracted (Polak et al., 1990). Possible fixatives include paraformaldehyde, formalin, or paraffin embedding. Cells must be treated with detergent or proteinases such as triton and RNAse-free proteinase K in order to permeabilize the membranes. The probe must have sufficient access into the cells so that it can bind with the target. The degree to which the cells are permeabilized affects the degree of specificity of ISH (Immuno-ISH, http://home.no.net/immuno/).
A variety of probes can be developed for ISH, depending upon the desired binding sensitivity. For DNA ISH, probes include double-stranded DNA, single-stranded DNA, and synthetic oligodeoxyribonucleotides. For RNA ISH, the probe can be single-stranded complementary RNA, called a riboprobe, that is synthesized by reverse cloning (Polak et al., 1990). Double-stranded DNA probes can be produced by PCR or replicated in bacteria. Double-stranded DNA probes must be denatured before hybridization and are often less sensitive because the single strands of DNA have a tendency to reanneal with each other. Single stranded DNA probes can be produced by PCR or reverse transcription of RNA. Oligonucleotide probes are synthetically produced and are relatively small (20-40 basepairs). Oligonucleotide probes can fit easilty through permeabilized cell membranes and can be designed to be very specific. Complementary RNA probes can be produced from RNA. RNA-RNA interactions are very stable and resistent to RNAses, however before hybridization they are difficult to work with (Power and Versatility of ISH, http://martin.parasitology.mcgill.ca/insituhybridization/INDEX.HTM).
Once the probe is developed, it must be labeled so that the location of the target sequence can be detected. Probes can be labeled directly, where the reporter molecule is directly attached to the probe, or indirectly, where a specific antibody or labeled binding protein is used to detect another molecule that is attached to the probe sequence. Radioactively labeled probes were originally used for ISH and continue to be used because they can be synthesized and incorporated into the DNA or RNA easily and autoradiography is relatively sensitive (Polak et al., 1990). However, radioactively labeled probes have a limited shelf life and require additional safety procedures and autoradiography can take several days. Non-radioactively labeled probes (fluorescently labeled probes) are very popular, as the procedures are readily available and less time consuming (Immuno-ISH, http://home.no.net/immuno/). Typical radioactively-labeled probes use 32P or 35S isotopes. Fluorescin and Rhodamine can be used for direct fluorescently labeled probes, and biotin and digoxigenin can be used for indirect fluorescence labeling. Fluorescent labeling can allow two or more different probes to be visualized at the same time because of color differences (Power and Versatility of ISH, http://martin.parasitology.mcgill.ca/insituhybridization/INDEX.HTM).
The degree of specificity to which the probe hybridizes to the target sequence can be controlled by the design of the probe and the conditions of the buffer solution, including temperature, pH, and salt concentration. “High stringency” conditions will only allow hybridization of probes with very similar homology to the target sequence, while “low stringency” conditions will allow a probe to bind with less specificity. Hybridization mixtures usually have a small volume (about 10-20 ul total) with 50% formamide and hybridization typically occurs between 37-60 degrees Celsius (Polak et al., 1990).
Several different techniques can be used to view where the probe has hybridized with the sequence of interest. Light field microscopy is most common and can be used for radioactively labeled probes or probes labeled with peroxidase or alkaline phosphatase. Fluorescent microscopes are used to view fluorescently labeled probes; the UV light excites the fluorescent die so that it can be detected through the microscope. Digital imaging systems are also used and can process the images and do quantitative measurements (Power and Versatility of ISH, http://martin.parasitology.mcgill.ca/insituhybridization/INDEX.HTM). See Figure 2 for an example of a FISH image.
Figure 2. FISH performed on chromosomes from a human peripheral blood lymphocyte. Chromosome 2 pair is labeled green and the Chromosome 4 pair is labeled orange. This image shows an example of how FISH can be used to identify specific pairs of chromosomes. (Permission pending for image found at http://www1.na.infn.it/wnucl/lines/radi/fish2.html.)
Like any good experiment, controls must also be developed for ISH to make sure that the probe is specific and binds only to the sequence of interest and that all steps of the procedure have been carried out properly. Controls for RNA In Situ Hybridization can include a poly-T tail to determine how strong of a signal is being detected (Power and Versatility of ISH, http://martin.parasitology.mcgill.ca/insituhybridization/INDEX.HTM). Northern and Southern blots can also be used to determine the strength of the signal for RNA and DNA. For apositive control for RNA, a probe for a constituitively expressed gene such as actin will demonstrate the procedure is working correctly. For a positive control for DNA, a probe should be synthesized for a highly repeated sequence (Leitch et al., 1994).
Applications of ISH
ISH is used in medical research and diagnosis, biology, pathology, and plant breeding. ISH can be modified in a variety of ways depending upon the material being analyzed. Some of ISH applications include determination of chromosome structure, function, and evolution, chromosomal gene mapping, expression of genes, localization of viral DNA sequences, diagnosis of viral diseases, and localization of oncogenes, sex determination. The uses and different approaches for ISH continue to increase, thus impacting many different research fields. New labeling techniques for probes, new detection systems, and advanced computer software increase the availability and efficiency of ISH (Leitch et al., 1994).
ISF was first introduced in 1969 independently by Gall and Pardue (1969), Buongiorno-Nardelli and Amaldi (1969), and John et al. (1969). Radioactively labeled RNA was used to detect DNA in vivo. Tritium was incorporated into proliferating cells in order to label the newly synthesized nucleic acids. While the labeling techniques and probe design have changed slightly, the basic technique of ISH remains the same today (Polak et al., 1990).
This site provides a good and clear overall description of In Situ Hybridization. It also has a cool slide show.
This site provides detailed information about ISH, along with information about immunofluorescence and ISH procedures.
This site gives several sample protocols for ISH.
This site talks about FISH involved with clinical testing.
Glockner, Frank Oliver, 2002. Fluorescence In Situ Hybridization. <http://www.mpi-bremen.de/molecol/fog/fish1.html >. Accessed 2003 Feb. 18.
Immunohistochemistry-In Situ Hybridization. In Situ Hybridization. <http://home.no.net/immuno/>. Accessed 2003 Feb. 16.
Leitch, A. R. et al. 1994. In Situ Hybridization. BIOS Scientific Publishers Limited, Oxford, pp. 1-33.
Polak, Julia M. and James O’D McGee. 1990. In Situ Hybridization: Principles and Practice. Oxford University Press, Oxford, pp. 10-30.
The Power and Versatility of In Situ Hybridization. In Situ Hybridization Lecture. < http://martin.parasitology.mcgill.ca/insituhybridization/INDEX.HTM>. Accessed 2003 Feb. 16.
Radiation Biophysics Laboratory. Flourescence In Situ Hybridization. <http://www1.na.infn.it/wnucl/lines/radi/fish2.html >. Accessed Feb. 18, 2003.
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