Northern blot hybridization detects mRNA levels and allows direct comparison of multiple samples. mRNA is extracted, separated by size, and exposed to radiolabelled probes. It can show gene expression patterns between tissue types and cellular responses to stimuli, providing insight into unknown proteins and responses.
Northern blot hybridization is a technique used to detect the levels of messenger RNA (mRNA) in a sample and to quantify the amount of mRNA present. While there are other, more powerful techniques, the Northern blot remains the standard due to its simplicity and ease of use. The Northern blotting technique also allows direct comparison of multiple samples in a single experiment.
Messenger RNA is a type of nucleic acid that is produced when a gene is expressed. An activated and functioning gene 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 matrix of proteins that the cell is producing.
In a Northern blot, mRNA is extracted from cells and passed through a denaturing agarose gel electrophoresis, which separates the mRNA in the sample based on size. Samples of mRNA molecules of known sizes are run simultaneously to provide a control. Once electrophoresis is complete, the samples are transferred and immobilized onto a solid membrane, typically made from a material such as nylon. The samples are then exposed to radiolabelled probes for several hours before analysis. During the assay, mRNA that has hybridized with a radiolabelled probe can be clearly detected by 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 tissue and organ types can be compared and cellular responses to stress, infection and many other stimuli can be examined. By observing the 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 the 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 can show that the expression of an unknown protein increases in response to the presence of a specific toxin. One could therefore theorize that the protein is involved in reducing toxicity, perhaps by transporting the chemical out of the cell or by converting it into a metabolite. If known proteins are under scrutiny, documenting changes in protein expression can provide insight into the response itself. If the expression of a known molecular transporter were increased, for example, it could be confirmed that the cellular response to the toxin is to transport the toxin out of the cell.
Protect your devices with Threat Protection by NordVPN
