NORTHERN BLOTTING

History of Northern blotting[1]

• Is one type of blotting for the RNA detection with labeled DNA probes.
• It was developed in 1977 by Alwine et al. Stanford University.
• It was named after the Southern blot technique which blots for DNA and
was invented by Edwin M. Southern in 1975.

Background information of Northern

Blotting Technique[1]
• Northern analysis despite its age in the high tech world of polymerase
chain reaction (PCR), nuclease protection assays (RPAs) and microarrays,
is still the gold-standard for the detection and quantitation of mRNA levels.
• The reason is it allows a direct comparison of the messenger RNA (mRNA)
abundance (amount of mRNA present in sample) between samples on a
single membrane.
• The main difference of Northern blotting from other techniques is that RNA
is the factor that used to be detected.
• RNA is separated out by:
 RNA gel electrophoresis (usually argarose)
 Subsequent transfer to membrane
 Hybridization with probe
 Detection
• For the hybridization probes it may be DNA or RNA in northern blotting
similar to Southern Blotting.
• A variant of the procedure known as the reverse northern blot was
occasionally (although infrequently) used.
• In this procedure, the substrate nucleic acid (that is affixed to the
membrane) is a collection of isolated DNA fragments, and the probe is
RNA extracted from a tissue and radioactively labelled.
• The used of DNA microarray have come into widespread use in late 1990
and early 2000 is akin to the reverse procedure. They involve the use the
use of isolated DNA fragment affixed to a substrate, and hybridization with
a probe made from cellular RNA.
• Thus the reverse procedure, though originally uncommon, enabled the
one-at-a-time study of gene expression using northern analysis to evolve
into gene expression profiling, in which many (possibly all) of the genes in
an organism
 Diagram of western blot procedure [1]

Northern blot principle[1]

1. RNA isolation
2. Gel electrophoresis of RNA for separation
3. Transfer to membrane (usually positively charged nylon as RNA is
negaticely charged)
4. Cross-linking of RNA to membrane (usually by UV-crosslinking or chemical
means)
5. Hybridization
6. Detection

Procedure [2]
1. RNA is isolated from several
biological samples (e.g.
various tissues, various
developmental stages of
same tissue etc.)
* RNA is more susceptible to
degradation than DNA.
 2. Sample’s are loaded on
gel and the RNA samples are
separated according to their
size on an agarose gel .
 The resulting gel following
after the electrophoresis run.

1. The gel is then blotted on a

nylon membrane or a
nitrocellulose filter paper by
creating the sandwich
arrangement.

Diagram of blotting sandwich [4]

2. The membrane is placed in a

dish containing hybridization
buffer with a labeled probe.

• Thus, it will hybridize to the

RNA on the blot that
corresponds to the sequence
of interest The probe can be either ss-DNA or
RNA
 1. The membrane is washed to
remove unbound probe.

Access probe is removed

2. The labeled probe is detected

via autoradiography or via a
chemiluminescence reaction
(if a chemically labeled probe
is used). In both cases this
results in the formation of a
dark band on an X-ray film.
• Now the expression patterns
of the sequence of interest in
the different samples can be
compared.

*Improvement in sensitivity of this method can be achieved by

using high specific activity antisense RNA probes (step 4),
optimized hybridization buffer (step 4), and positively charged
nylon membrane (step 3) [2]
*RNA ISOLATION
This part of the Northern Blot is an important step because of high quality, intact RNA
needs to be obtained. There are several ways of the isolation can be performed.
However, common attributes include cellular lysis and membrane disruption, inhibition of
ribonuclease activity, deproteinization and recovery of the intact RNA. [3]

Applications of the northern blot[1]

Have been superseded in most areas by Real Time PCR and microarray
approaches but it is not often used in clinical and diagnostic purposes.
The northern blot principle and its variations are used however in molecular
biology research to:
• A gold-standard for the direct study of gene expression at the level of
mRNA (messenger RNA transcript)
• Detection of mRNA transcript size
• Study of RNA degradation
• Study of RNA splicing (can detect alternatively spliced transcript)
• Study RNA half life
 • Study IRES (internal ribosomal entry site) –to remove possibility or RNA
digestion vs second cistron translation
• Often used to confirm and check transgenic / knockout mice (animals)
*transgenic – containing genetic material into which DNA from a different
organism has been artificially introduced.

Advantages of northern blotting[1]

• Widely accepted and well regarded method
• It is a straight-forward method
• Often it is used as a confirmation or check
• Often a gold-standard
• It is a versatile principle which allow the usage of many types of probes
(vs Real Time PCR) including : radiolabeled and non-radiolabeled, in vitro
transcribed RNA and even oligonucleotides such as primers.
• Sequences with even partial homology, unlike real time PCR or methods
can be used as hybridization probes (sequence from different species for
homology analysis, or even genomic fragments can be used)

Disadvantages of northern blotting

technique[1]
• The whole of northern blotting process takes a long time, from the
preparation through to detection.
• The standard of northern blot is less sensitive compared to the nuclease
protection assays and Real Time PCR. The sensitivity may be increased
with the use of nylon positively-charged, use of a highly specific antisense
probe.