Northern Blotting - Research Paper Example

Northern blotting is a technique that detects the messenger RNA (mRNA) in a sample that is used for studying gene expression. The mRNA is produced during the transcription of a gene and this serves as a template for translation into a protein. The northern blotting technique can be used to study the proteins expressed in specific tissues, which can provide an insight to their physiological function. In addition, the technique is also used to determine the factors that regulate gene expression such as nutritional, hormonal or environmental agents (Trayhurn).

A group of three scientists at the Stanford University, James Alwine, David Kamp and George Stark developed this technique in the year 1977. As by then the southern blotting technique, named after its founder, was already in existence this technique was named the northern blotting without any reference to those who developed the technique (Trayhurn; Technique of Northern Blotting; Northern Blot). The basic principle of the technique is extraction of the mRNA from the sample which is detected on a membrane using a hybridization probe that has a base sequence complimentary to part or the entire mRNA.

The process involves isolating the mRNA from the total RNA sample extracted from the tissue and this step is carried out using an oligo-T chromatography column in which the oligo-T bind to the poly-A tail of the mRNA. The RNA is then separated based on its molecular size by running it through an agarose gel electrophoresis. The RNAs separated in the gel is then blotted on to a nylon membrane that is usually positively charged and has a high affinity to bind nucleic acids. The northern blotting technique particularly refers to this step of the procedure where the mRNA is blotted on to a membrane either through vacuum or capillary method.

This blot is an exact copy of the separated gel in the agarose membrane. This transfer is done in order to enable better hybridization of the probes on to the specific mRNAs as it is difficult for the probes to penetrate the gel compared to the nylon membrane. Once the RNA is blotted on to the nylon membrane, it is immobilized to the membrane by either using heat or exposing the nylon membrane to UV light. This will result in the formation of covalent linkages between the mRNA and the nylon membrane, which will help prevent its loss during the subsequent washing process.

This is followed by hybridization to a probe which is usually a complimentary DNA (cDNA) that has specific nucleotides that will bind to the target mRNA. The probes are radiolabelled and the radioactivity is later detected. Chemiluminescence or fluorescence techniques are the non-radioactive techniques that are also being used. The signals from the hybridization process are collected with an x-ray film which can also be quantified using a densitometer. The gene product is then determined using the microarray technique or RT-PCR method (Trayhurn; Technique of Northern Blotting).

Applications of Northern Blotting The northern blotting technique remains the gold standard to study the expression of genes at the mRNA level in tissues and organs and determine the protein produced by the gene (Technique of Northern Blotting; Northern Blot). With regard to the specific study of RNAs, the technique can be employed to detect the transcript size of mRNA, study the degradation of RNA, splicing transcripts that are produced, RNA half-life, and study the internal ribosomal entry site, confirmation of transgenic mice (Northern Blot).

The function of unknown proteins can also be detected using this technique. In case of cancerous tissues, the over-expression of oncogenes and the reduction in the expression of tumor suppressor genes can be detected using this technique (Technique of Northern Blotting). Variant method A variation of the general northern blotting technique is the reverse method in which is used only quite rarely. In this method, isolated DNA fragments are linked to the membrane while the RNA isolated from the tissues and radioactively labeled acts as the probe.

The DNA microarray technique, which is now being used often, is based on this reverse technique (Northern Blot). Advantages and disadvantages of the technique The technique has several advantages such as being a straight-forward method with a versatile protocol that allows the use of both radiolabelled and non-radiolabelled probes; sequences which have a lesser homology can also serve as probes as the method is sensitive. In addition, both the quantity and quality of the RNA can be measured on the gel before beginning the blotting process.

The membranes also have the advantage of a longer storage time and they can be probed again. However, the technique suffers from certain disadvantages such as possible safety hazards when radioactivity is used, time-consuming, interference by RNASes, problems with the use of multiple probes as in such cases probes must be stripped off the membrane in order to be detected with a different probe and the process is both tedious and has its own limitations. To avoid radioactive hazards, non-radioactive agents need to be used and to avoid the effect of RNases glassware should be sterilized and RNase inhibitors need to be included during the procedure.

This technique can be used only on a small number of genes unlike the more refined techniques such as microarrays in which can include multiple genes. In addition, small changes in the gene expression are also observed in the technique that is not present when microarrays are carried out. Thus while the technique still remains the gold standard for studying gene expression, more refined variants of the technique are increasingly used in molecular biology studies (Technique of Northern Blotting; Northern Blot).

Work Cited Trayhurn, Paul. “Northern Blotting.” Proceedings of the Nutrition Society 55 (1996): 583-589. Print “Technique of Northern Blotting.” Biotecharticles.com. Biotech Articles, 2 Sept. 2010. Web. 28 Apr. 2012. http://www.biotecharticles.com/Biotechnology-products-Article/Technique-of-Northern-Blotting-382.html “Northern Blot.” Molecularstation.com. Molecular Station, n.d. Web. 28 Apr. 2012. http://www.molecularstation.com/rna/northern-blot/