In-Situ Hybridization
What Would You Use it For?
Whole mount in-situ hybridization is used to view the location of nucleic acids in situ (ie. in their native location in the cell or nucleus).
How Would You Use It?
For viewing the location of RNA in the cell:
The tissue is gently fixed so as not to disrupt the dsDNA. Tissues are then hybridized to a chemically or radioactively labeled RNA or DNA probe that is complimentary (or anti-sense) to the desired mRNA of interest. The chemical or radioactive probe can then be detected in the tissue by antibodies to the chemical label or by autoradiography. When using the chemically labeled probe the antibody may be fluorescently labeled or can have an enzyme associated with it that when exposed to substrate generates a colored precipitate so that localization in cells is easy.
For viewing DNA in the nucleus:
Chromosomes are briefly exposed to high pH to disrupt the DNA base pairs followed by hybridization and visualization as above.
Pros and Cons:
-This technique allows you to visualize the location of your nucleic acid in its cellular context in contrast to cell homogenates which only allow detection of the presence or absence of a nucleic acid.
-Information can be inferred about protein and trascription regulation by the presence or absence of mRNA in a cell when compared to the protein expression pattern of that gene.
-This technique can be used on sectioned tissues as well as on whole embryos or tissues. -Whole embryo techniques have been extremely useful in understanding patterning during embryonic development.
-If the mRNA of your desired gene is not in abundance it may be hard to detect a signal from in-situ probing.
Controls you should run and why:
-When detecting mRNA you use an anti-sense RNA probe which is complimentary to the mRNA sequence you are looking for in the cell. You should also perform a sense strand control in parallel. The sense control will give you a background signal indicating the level of non-specific interactions.
-When looking for DNA location in the cell both the "sense" and "anti-sense" signals should be detectable, and should be localized similarly in your samples as the DNA is only denatured and has not changed its location in the cell.
Result Interpretations:
This technique allows you to view the location of mRNA and DNA in its native location. In the case of RNA this information only along with data from other types of experiments will allow you to infer information about regulation of transcription or translation. Localization of DNA again only allows you to localize your gene of interest to a specific location on a chromosome or will allow you to see if the gene is present in multiple copies in the cell but will not give you any information about expression or regulation.
Editor(s): John Bracht
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