Northern Blot

The Northern blot is a molecular biological method for transferring ( blotting ) of the separated in gel electrophoresis RNA to a membrane ( diazobenzyloxymethyl ( DBM) paper or under certain conditions, nitrocellulose or nylon). On the membrane, the specific labeling of RNA sequences is possible because of the hybridization with the complementary probes.

The name Northern blot was chosen in allusion to the name of the Southern blot method developed by Edwin Southern ( in the DNA rather than RNA is blotted ). The name is an allusion, as the cardinal points have nothing directly to do with the process.

The Northern blot method was introduced in 1977 by James Alwine, James Kemp and George R. Stark at Stanford University for diazobenzyloxymethyl ( DBM) - paper, and in 1980 switched from Patricia Thomas as in the simpler Southern blotting to nitrocellulose. In 1979, G. Stark another Blottingmethode ( for the separation of proteins ), which he described as a Western blot in continuation of the word game.

Application

The method of northern blotting is used, for example, to compare the mRNA coding for a protein of a mutated organism to that of a "normal" body. A trial might look like this:

It is tissue samples in containing the protein, or would have to occur both from mutants as well as from normal individuals (as a control ) and closes the cells in an in detergent highly concentrated solution, which inactivates nucleases so that these nucleic acids can not attack. After the proteins were separated on the liquid have been removed such as by denaturation, and repeated phenol extractions, as well as smaller molecules by precipitation in alcohol, RNA and DNA are separated by their different solubilities in alcohol. Contamination of DNA can now, for example, be degraded by the highly specific enzyme DNase. mRNAs can be separated from other RNA by retention on a chromatographic column, which binds specifically to poly (A ) tail of the mRNAs. Now first the intact and purified mRNA molecules are separated by gel electrophoresis according to their size. So that the RNA molecules are available for detection, transmits ( blotting) to. The RNA with the corresponding band pattern on a sheet of nitrocellulose paper In this membrane, a labeled DNA or RNA probe is added the sequence of which corresponds to a portion of the mRNA to be detected. The RNA molecules on the membrane that hybridize with the labeled probe, can now be detected by autoradiography or chemically. The size of the RNA molecules in each band can be determined by comparison with RNA molecules of known size ( RNA standards ), which is allowed to run in parallel with the experimental samples. Now you can see if and how the coding for proteins of the mutant mRNA is different from that of the normal individual.