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In Situ Hybridisation Page
In situ hybridisation is a method for detecting messenger RNA (mRNA) or DNA in cells or thin sections (slices) of tissue, usually mounted on glass microscope slides, so that the location of the target molecule can be seen in individual cells. This technique is very valuable in the investigation of disease (pathology) and is widely used in many laboratories all over the world. The diagrams below illustrate this:
It is possible to detect DNA or RNA using specific probes made themselves from DNA or RNA, because of the unique way in which single strands of RNA or DNA will bind to an opposite strand.
A probe is made by amplifying a specific DNA sequence using the polymerase chain reaction, then this sequence is placed in a plasmid vector and used to transform bacteria. This produces a large amount of DNA which can be digested with specific enzymes. Finally, the sequence is transcribed using RNA polymerases and the building blocks of RNA. A label is introduced during transcription so that the probe can be detected later. This is known as a complementary RNA probe or riboprobe.
The labelled probe is hybridised to a section of tissue or cells stuck onto a glass slide. The binding of probe to the target RNA in the cells is very specific. The label is then visualised by a variety of methods.
Examples of in situhybridisation results are shown below.
Prolactin mRNA in pituitary cells (non-radioactive in situ hybridisation).
Prolactin mRNA in pituitary cells detected with a fluorescent probe (green). The nuclei are stained blue.
Combined in situhybridisation and immunocytochemistry on the same tissue section. Prolactin mRNA in one type of pituitary cells detected with a non-radioactive probe. Neighbouring cells of another type containing growth hormone (GH) have been stained using immunocytochemistry, a staining method using specific antibodies to identify proteins in cells. A) Prolactin mRNA is stained black, GH protein is stained red. B) Prolactin mRNA is stained red, GH protein is stained brown.
Thyroid-stimulating hormone (TSH) mRNA in another type of pituitary cells detected with a radioactive probe and a photographic method called autoradiography (black grains). TSH protein is shown in the same cells using immunocytochemistry (orange/brown staining).
TFF3 mRNA in intestinal goblet cells detected with a digoxigenin-labelled riboprobe.
TFF3 is otherwise known as intestinal trefoil factor. In this picture, the mRNA is labelled brown and the mucin products of goblet cells are stained blue with alcian blue/PAS.
Bacteria in human tissue, fluorescent in situ hybridisation.
Bacteria in human placenta, fluorescent in situ hybridisation.
Bacteria in human tissue, fluorescent in situ hybridisation.
X and Y Chromosomes detected in human cells using fluorescent in situ hybridisation (FISH).
See references to some of my published papers on in situ
hybridisation
References page
My work has been supported by medical research charities.
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