In situ hybridisation

• In situ hybridisation (ISH) is the detection of a target DNA or RNA sequence in a tissue section using a labelled nucleic acid probe. It is still the only hybridisation technique which allows cellular and subcellular localisation of the target.
• The ISH protocols in this manual have been designed for use with high sensitivity, radio-labelled RNA probes and optimised for the detection of low abundance messenger RNAs (mRNAs) in adult rodent tissues. (G. Niedobitek and H. Herbst (1991) Applications of in situ hybridisation International Review of Experimental Pathology. Ed Richter, G.W. Solez, K. 32:1 - 56. Harcourt Brace Jovanovitch)
• To identify the site of mRNA expression, the mRNA must be chemically cross-linked or 'fixed' in place prior to hybridisation. It is fixed in its undenatured state, still intimately associated with normal tissue components. Consequently, not all of the mRNA within the tissue section is available to form probe : target hybrids. This effectively reduces the maximum hybridisation signal that can be obtained. Also, by interacting with the RNA probe, normal tissue components create a non-specific background which is peculiar to ISH and not encountered with standard filter or solution hybridisations.
• As with any hybridisation, the balance between specific signal and non-specific background, the signal : noise (S/N) ratio, determines whether a particular target mRNA can be detected by ISH. The signal from abundant mRNAs is easily detected over high backgrounds. However, even low backgrounds can obscure the small signal from low abundance mRNAs and a good S/N ratio is crucial for their detection.
• These protocols have evolved from those of Dr. Paul Senior. I am also grateful to Dr. Jenny Penschow and Ms Lynne Hartley for their advice and for sharing their expertise.

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