Northern blot

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The northern blot is a technique used in molecular biology research to study gene expression by detection of RNA (or isolated mRNA) in a sample.[1][2]

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.[3] Northern blotting involves the use of electrophoresis to separate RNA samples by size, and detection with a hybridization probe complementary to part of or the entire target sequence. 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.[4] The northern blot technique was developed in 1977 by James Alwine, David Kemp, and George Stark at Stanford University.[5] Northern blotting takes its name from its similarity to the first blotting technique, the Southern blot, named for biologist Edwin Southern.[1] The major difference is that RNA, rather than DNA, is analyzed in the Northern blot.[6]

Contents

Procedure

A general blotting procedure[4] 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 a poly(A) tail.[7][8] RNA samples are then separated by gel electrophoresis. Since the gels are fragile and the probes are unable to enter the matrix, the RNA samples, now separated 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.[9] 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.[10] The membrane is washed to ensure that the probe has bound specifically and to avoid background signals from arising. The hybrid signals are then detected by X-ray film and can be quantified by densitometry. To create controls for comparison in a northern blot, samples not displaying the gene product of interest can be used after determination by microarrays or RT-PCR.[10]

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