What is Northern Blotting?
Northern blotting is a technique used in molecular biology with the principal aim being to measure a specific messenger RNA (mRNA). The technique is a modified version of Southern Blotting. The techniques are essential in molecular biology for the detection of nucleic acids extracted from different types of biological samples.
Why should we Measure an mRNA?
The main reason for measuring is to determine which tissues express a particular gene to give an indication of the physiological function of the encoded protein for example the discovery that the obese gene is expressed in white adipose tissue an indication that the protein product (Leptin) acts as a signal for the size of the fat depots
Secondly, the mRNA, is measured to determine the factors that regulate the expression of a given gene such as nutritional, hormonal, or environmental.
Techniques for measuring mRNA,
There are 3 techniques used
RNase protection assay
The reverse transcriptase-polymerase chain reaction
Principles of Northern Blotting
The principle is similar to that in Southern Blotting the only difference is the probes used in detection as northern blotting detects RNA sequence providing information about the length of the RNA sequence and the presence of variations in the sequence, in addition, quantification of the RNA sequence. The principle is based on the transfer of biomolecules from one membrane to another. RNA is separated by size and detected on a membrane using a hybridization probe. The process involves the transfer of RNA from a gel to a membrane. RNA is extracted from a tissue using chaotropic agents such as guanidinium isothiocyanate to disrupt cells and denture proteins and solubilize RNA. Alternatively, a separate step may be included for the isolation of mRNA, from total RNA, this increases the sensitivity since only a small percentage of the total RNA is extracted from tissue in mRNA.
Isolation of mRNA, from total RNA, uses a poly-A+ selection procedure that involves an oligo-T column or beads primed with oligo-T, to bind to the ply-A+ tail of mRNA. The extracted RNA species are separated based on molecular size through agarose-gel electrophoresis. Then blotting is done onto nylon membranes, particularly positively charged membranes due to their high-binding capacity for nucleic acids and their greater robustness in handling. Following blotting, the RNA is immobilized on the membrane by baking in an oven or by exposure to U.V light resulting in the covalent linkage of RNA to the membrane. This prevents the nucleic acid from being washed away during the subsequent processing. The hybridization probe is then prepared and the probe hybridized with the membrane. Later, the post-hybridization is strangely washed to ensure the probe is bound specifically to the target mRNA. Also, that there is negligible non-specific binding to other mRNA. or the nylon membrane itself. Here the hybridization signals are detected by the film and quantified where required
Northern Blot is simple and flexible. It is the only method that provides information about the size of the RNA. Also, the technique is specific. It is the technique of choice in the characterization of mRNA, that has been used successfully to compare the relative abundance of a particular gene expressed in cells subjected to different experimental and physiological conditions. Furthermore, it has been used to study the roles of certain proteins in the development and suppression of cancerous growth. The technique can also provide useful information about the function of a certain gene and in the study of the alternative splicing of the different gene products and the study of the abnormal gene and genetic disorders.