Northern Blotting:
Northern blotting is an RNA blotting technique which was
developed in 1977 by Alwine et al. at Stanford University. It was named after the Southern blot
technique which blots for DNA, invented by Edwin M. Southern in 1975. Northern analysis
is still the gold-standard for the detection and quantization of mRNA levels. This
is because northern blot analysis allows a direct comparison of the messenger
RNA abundance between samples on a single membrane. I n northern blot the
main difference between the other blotting techniques is that RNA is the
factor being detected. Also, due to the fact that RNA is usually
single-stranded, it creates complex secondary structures which affect its
migration and hence denaturing conditions are used to run the gels. RNA is
separated out by RNA gel electrophoresis (usually agarose gel electrophoresis),
subsequent transfer to membrane, and hybridization with probe, and finally
detection rad.
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. It makes 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. It also 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.
Procedure:
l A
general blotting procedure starts with extraction of total RNA from a
homogenized tissue sample.
l The
mRNA can then be isolated through the use of oligo (dT) cellulose
chromatography to maintain only those RNAs with a poly(A) tail.
l RNA samples are then separated by gel
electrophoresis.
l 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.
l 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.
l 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
l .Once
the RNA has been transferred to the membrane it is immobilized through covalent
linkage to the membrane by UV light or heat.
l After
a probe has been labeled, it is hybridized to the RNA on the membrane
l Experimental
conditions that can affect the efficiency and specificity of hybridization
include ionic strength, viscosity, duplex length, mismatched base pairs, and
base composition.
The hybrid signals
are then detected by X-ray film and can be quantified by densitometry.
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