NORTHERN BLOTTTING

NORTHERN BLOTTTING : It is developed by James Alwine, David kemp, and George stark at standford Uninversity. Nothern blotting. Northern blotting takes its name from its similarity to the first blotting technique, the Southern Blot, named for biologist Edwin Southern The major difference is that RNA, is analyzed in the Northern blot while DNA in Southern Blot. With northern blotting it is possible to observe cellular control over structure and function by determining the particular gene expression levels during differentiation, morphogenesis, as well as abnormal or diseased conditions. Northern blotting involves the use of electrophoresis to separate RNA samples by size, and detection with a hybridization probe. The term 'northern blot' actually refers specifically to the capillary transfer of RNA from the electrophoresis gel to the blotting membrane, however the entire process is commonly referred to as northern blotting.
 
PROCEDURE :A general blotting procedure starts with extraction of total RNA from a homogenized tissue sample. The mRNA can then be isolated through the use of oligo (dT) cellulose chromatography to maintain only those RNAs with poly (A)tail RNA samples are then separated by gel electrophoresis by size, are transferred to a nylon membrane through a capillary or vacuum blotting system.
A nylon membrane with a positive charge is the most effective for use in northern blotting since the negatively charged nucleic acids have a high affinity for them. The transfer buffer used for the blotting usually contains formamide because it lowers the annealing temperature of the probe-RNA interaction preventing RNA degradation by high temperatures. Once the RNA has been transferred to the membrane it is immobilized through covalent linkage to the membrane by UV light or heat. After a probe has been labeled, it is hybridized to the RNA on the membrane. Experimental conditions that can affect the efficiency and specificity of hybridization include ionic strength, viscosity, duplex length, mismatched base pairs, and base composition. The membrane is washed to ensure that the probe has bound to only specifically RNA and to avoid background signals from arising. The hybrid signals are then detected by X-ray film and can be quantified by densitometry.
 
APPLICATION OF NOTHERN BLOT :
Northern blotting allows one to observe a particular gene's expression pattern between tissues, organs, developmental stages, environmental stress levels, pathogen infection, and over the course of treatment.
The technique has been used to show overexpression of oncogenes and downregulation of tumor-suppressor genes in cancerous cells when compared to 'normal' tissue,
It is also used in gene expression in the rejection of transplanted organs.
If an upregulated gene is observed by an abundance of mRNA on the northern blot the sample can then be sequenced to determine if the gene is known to researchers or if it is a novel finding.
The expression patterns obtained under given conditions can provide insight into the function of that gene.
RNA is first separated by size, if only one probe type is used variance in the level of each band on the membrane can provide insight into the size of the product, suggesting alternative splice products of the same gene or repetitive sequence motifs. The variance in size of a gene product can also indicate deletions or errors in transcript processing, by altering the probe target used along the known sequence it is possible to determine which region of the RNA is missing.

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