Northern blotting is a technique of molecular biology which is used to detect the RNA in a sample in the study of gene expression. It is similar to the process of southern blotting in which DNA molecule is isolated to study the gene expression. A suitable DNA probe is used for the detection of particular mRNA.
In 1977, James Alwine, David Kamp and George Stark at the Stanford University developed the technique of northern blotting. The name of this technique was chosen because of the technique which was already in use that is Southern Blotting.
Following steps are involved in the procedure;
1) Process of northern blotting starts with the extraction of RNA from a tissue sample.
2) mRNA is isolated from this RNA by using the oligo cellulose chromatography so that only those mRNAs can be taken which have poly A tail.
3) The technique of gel electrophoresis is used to separate the RNA samples according to their size. Due to the fragility of gel, samples cannot enter the matrix. For this purpose, nylon membrane is used in which the samples are inserted through capillary or vacuum blotting system.
4) When the RNA sample transfer is complete on nylon membrane, it is made immobilized with covalent linkage by using the hat or ultraviolet light.
5) As described earlier that DNA probe is used in the process, so when the RNA sample is fixed on the nylon membrane, it is hybridized with the tagged probes.
6) When the probes are hybridized, now the membrane is washed to see the perfect hybridization with the sample.
7) Signals of hybridization are detected with x-ray and they can also be quantified by densitometry.
8) Microarrays or RT-PCR determine the gene product.
Uses of Northern Blotting:-
Northern blotting is a very useful technique of molecular biology as it allows the gene expression pattern between organs, tissues, infection of pathogens and other treatments. When the comparison of normal tissues and cancerous tissues is concerned, then this technique is of great importance because it enables the oncogenes to show overexpression and also the downregulation of tumor suppressor genes. It also helps the scientists to determine that what type of proteins is produced by examining different expression levels of mRNA. Northern blotting can also enable the scientists to know the function of unknown proteins.
Advantages of Northern Blotting:-
Northern blotting enables the scientists to detect the size of RNA. It also allows them to observe the alternate splice products, using the probes with partial homology. It is of great benefit in measuring the quality and quantity of RNA on the gel before the blotting technique starts. The membranes can be probed again once they are used in blotting and can be stored for many years.
Disadvantages of Northern Blotting:-
There are many techniques in biotechnology which can be used to analyze the expression of the genes like, Microarrays, RT-PCR and serial analysis. Northern blotting is also one of these techniques, which serves the same purpose. When this technique is applied for the gene expression, it shows small changes in the expression of the genes but if the same sample is passed through microarrays, no changes appear. Another disadvantage of northern blotting is that it is applied only on a small sample of the genes while microarrays technique can be applied on many genes.
Another disadvantage of northern blotting is that when the sample is provided, it can be degraded by RNases. To avoid this problem, glassware should be sterilized and RNase inhibitors should be used. The chemicals which are used in this technique can be harmful for the researcher's health because they contain formaldehyde, ethidium bromide, radioactive material and ultra violet light.
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