7.25C: Northern Blots
- Evaluate the applications of Northern Blots
The Northern blot is a technique used in molecular biology research to study gene expression in a sample, through detection of RNA (or isolated messenger RNA ). 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 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. The northern blot technique was developed in 1977 by James Alwine, David Kemp, and George Stark at Stanford University. 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, rather than DNA, is analyzed in the Northern blot.
A general blotting procedure starts with extraction of total RNA from a homogenized tissue sample or from cells. Eukaryotic mRNA can then be isolated through the use of oligo (dT) cellulose chromatography to isolate only those RNAs with a poly(A) tail. 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.
Key Points
- RNA (either total RNA or just mRNA) is separated by gel electrophoresis, usually an agarose gel. Because there are so many different RNA molecules on the gel, it usually appears as a smear rather than discrete bands.
- The RNA is transfered to a sheet of special blotting paper called nitrocellulose, though other types of paper, or membranes, can be used. The RNA molecules retain the same pattern of separation they had on the gel.
- The blot is incubated with a probe which is single-stranded DNA. This probe will form base pairs with its complementary RNA sequence and bind to form a double-stranded RNA-DNA molecule. The probe is either radioactive or has an enzyme bound to it.
Key Terms
- hybridization : The act of hybridizing, or the state of being hybridized.