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Northern Blotting - Research Paper Example

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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…
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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 Read More
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