Northern blot
The Northern Blot is a molecular biological method for transferring ( blotting ) the RNA separated in gel electrophoresis onto a membrane (diazobenzyloxymethyl (DBM) paper) or under certain conditions ( nitrocellulose or nylon ). Specific labeling of RNA sequences on the membrane is possible through hybridization with complementary gene probes .
The name Northern Blot was chosen as an allusion to the name of the Southern blot method developed by Edwin Southern (in which DNA is blotted instead of RNA ). The name is an allusion because the cardinal points have nothing to do with the procedure.
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 Patricia Thomas switched to nitrocellulose as in the simpler Southern Blot . In 1979, Stark developed another blotting method (for separating proteins), which he called Western Blot , a continuation of the pun . The combination of Western and Northern blots is called Northwestern blots .
application
The Northern blotting method is used, for example, to compare the mRNA coding for a protein of a mutated organism with that of a wild-type organism. A corresponding attempt could look like this:
Tissue samples are taken in which the protein occurs or should exist, both from the mutant and from the normal individual (as a control) and the cells are disrupted in a highly concentrated detergent solution that inactivates the nucleases so that they do not contain the nucleic acids can attack. After the superfluous proteins have been separated off, for example by denaturation and repeated phenol extractions, as well as smaller molecules have been removed by precipitation in alcohol, RNA and DNA are separated from one another due to their different solubilities in alcohol. Contamination of DNA can now e.g. B. be degraded by the highly specific enzyme DNase. mRNAs can be separated from other RNAs by retention on a chromatographic column that specifically binds the poly (A) tails of the mRNAs. Now the intact and purified mRNA molecules are first separated according to their size by gel electrophoresis . Thus, the RNA molecules are available for detection, blotted ( blotting ) to the RNA with the corresponding band pattern on a sheet of nitrocellulose paper. A labeled DNA or RNA probe, the sequence of which corresponds to a section of the mRNA to be detected, is placed on this membrane. The RNA molecules on the membrane that hybridize with the labeled probe can now be detected autoradiographically or chemically. The size of the RNA molecules in each band can be determined by comparison with RNA molecules of known size (RNA standards) that are run in parallel with the experimental samples. Now one can see whether and to what extent the mRNA coding for proteins of the mutant differs from that of the normal individual. Advantages of the method are the possibility of making statements about the size of the transcript, the quality of the RNA and the occurrence of isoforms. The relative amount of the detected transcript can be determined from the signal. The filters can also be hybridized with other samples. Alternative methods of detecting individual transcripts are the reverse transcriptase polymerase chain reaction (RT-PCR), the RNAse protection assay and in situ hybridization . Disadvantages are the high experimental effort and the low sensitivity.
Individual evidence
- ↑ James C. Alwine, David J. Kemp, George R. Stark : Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes. In: Proceedings of the National Academy of Sciences . Vol. 74, No. 12, 1973, pp. 5350-5354, PMID 414220 , PMC 431715 (free full text).
- ↑ Patricia S. Thomas: Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose . In: Proceedings of the National Academy of Sciences . Vol. 77, No. 9, 1980, pp. 5201-5205, PMID 6159641 , PMC 350025 (free full text).