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Northern blot hybridization is a technique which is used to detect messenger RNA (mRNA) levels in a sample, and to quantify the amount of mRNA which is present. Although other more powerful techniques do exist, northern blot remains the standard due to its simplicity and ease of use. The northern blotting technique also allows for direct comparison of multiple samples in a single experiment.
Messenger RNA is a type of nucleic acid which is produced when a gene is being expressed. A gene that is switched on and functional within a cell is transcribed into mRNA. The mRNA is then transported into the cytoplasm of the cell, where it is translated into a protein. Northern blot analysis of a cellular mRNA sample can therefore provide useful information about the array of proteins the cell is producing.
In a northern blot, mRNA is extracted from cells and run through a denaturing agarose gel electrophoresis, which allows mRNA in the sample to be separated according to size. Samples of mRNA molecules of known sizes are run at the same time to provide a control. Once electrophoresis is complete, the samples are transferred and immobilized on a solid membrane, typically made from a material such as nylon. The samples are then exposed to radiolabeled probes for several hours before analysis. During the analysis, mRNA that has hybridized to a radiolabeled probe can be clearly detected using x-ray.
The northern blotting technique allows for the observation of gene expression patterns between different tissue types. Messenger RNA extracted from cells of various different tissues and organ types can be compared, and cellular responses to stress, infection, and many other stimuli can be examined. By looking at differences in mRNA expression under different conditions, a scientist can determine what types of proteins a cell produces in response to certain stimuli. This can provide clues about functions of unknown proteins, or in the case of known proteins, information about cellular responses to the stimuli in question.
For example, a northern blot may show that the expression of an unknown protein increases in response to the presence of a specific toxin. It could then be theorized that the protein is involved in reducing toxicity, perhaps by transporting the chemical out of the cell or converting it into a metabolite. If known proteins are under examination, documenting changes in protein expression can provide information about the response itself. If expression of a known molecular transporter was increased, for example, it could be confirmed that the cellular response to the toxin is to transport the toxin out of the cell.
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