what is in situ hybridization?

Question

i would like to know some details please

Answer 1

Ick, I hate in situ!

But anyways, as you know, cells transcribe single stranded mRNA from the double stranded DNA of genes. If one could detect this ss mRNA, one could know which genes a cell was expressing, right? So, ISH is one way of detecting mRNAs.

The short version is that an antisense RNA is made in vitro and this is allowed to bind to the sense RNA in the tissue by base pairing. By incorporating radioactivity, or some other label, into the antisense probe the locations that contain the sense RNA one is interested in can be detected, and information can be inferred about gene expression.

i.e. if a doctor was curious about whether a tumor was expressing the estrogen receptor, an antisense RNA to the estrogen receptor could be made labeled with 33P. The probe could be put on tissue sections, unbound probe subsequently washed off, and the remaining radioactivity would mark the ER expressing cells.

"In situ" refers to the detection of the mRNA in tissue sections or similar preparations, not homogenized tissue.
"Hybridization" means that the antisense probes are binding the sense RNAs by means of base pairing that yields a double stranded RNA hybrid in the tissue.

This is a common technique and searching the pub med database will turn up tons of references, both technical descriptions and examples of the technique applied to generate data. There are even whole books on it, if you need a manual.

Answer 2

In Situ Hybridization

Answer 3

those two guys above gave some very good answers.
I used to do in situ hybridization on a routine basis to determine the location of genes on chromosomes. In my protocol, we labelled DNA with biotin-dUTP. The chromosomes were spread out and affixed onto a glass slide. Both the DNA in the chromosomes and the probe were denatured (made single-stranded.) The probe was incubated with the chromosomes so that the probe stuck (i.e. hybridized) to the complementary portion of the chromosome. this was followed by incuabtion with avidin-HRP. Avidin binds to biotin. The HRP can be detected by reaction with DAB, producing a brown precipitate at the point of hybridization. A related procedure utilizes fluorescent probes. This is called FISH (fluroescent in situ hybrization.) The use of in situ hybridization to locate genes on chromosomes has tapered off somewhat with the completion of numerous genome projects.

Answer 4

It means hybridization at the site. Typically it means hybridization of a radioactive DNA or RNA probe to a sample containing a tissue section or chromosomes immobilized on a slide. It can be used to locate the position of a given gene on a particular chromosome and to a particular region of that chromosome (short or long arm for example). It can also be used to detect RNA copies (gene transcripts) within a histological section.

Answer 5

It takes six seconds for the Yahoo Answers page to open for me to be able to click on the [add your answer] button. is my computer slow?

Answer 6

I'm curious as well