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Northern Blotting technique

Northern blotting is a molecular biology technique used to study gene expression by detecting specific RNA sequences. Developed in 1997, it involves isolating RNA, electrophoresis, transferring RNA to a membrane, hybridization with probes, and autoradiography for detection. This method has various applications, including studying gene expression conditions, oncogene behavior in cancer, and mRNA splicing.

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10 views5 pages

Northern Blotting technique

Northern blotting is a molecular biology technique used to study gene expression by detecting specific RNA sequences. Developed in 1997, it involves isolating RNA, electrophoresis, transferring RNA to a membrane, hybridization with probes, and autoradiography for detection. This method has various applications, including studying gene expression conditions, oncogene behavior in cancer, and mRNA splicing.

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ashwaijaz
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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Northern Blotting:

The northern blotting is a very useful technique in molecular biology. It is used to study gene
expression by detecting specific RNA sequences in a sample.

History of northern blotting:


Northern blotting was first developed by James Alwine and George Stark in 1997 at Stanford
university. It was named by the analysis of southern blotting to detect size and quantify RNA.

Purpose of Northern blotting


The term Northern blot is used for the capillary transfer of RNA from the electrophoresis gel
to the blotting membrane. It involves the use of electrophoresis to separate RNA samples
depend upon their size and detection with a hybridization probe. Actually it is mRNA which is
isolated and hybridized in Northern blots.

Steps in northern blotting:


There are following steps in the northern blotting

Step 1

Isolate RNA:
The first step is the separation of RNA. RNA can be isolated from several biological samples
such as from various tissues. RNA is more susceptible to degradation than DNA. For the purpose
to detect rare mRNA isolate the polymerase mRNA.

Step 2

Electrophoresis:
In the second step the samples are loaded on the agarose gel. This experiment is done on the
formaldehyde agarose gel. Because this gel prevents RNA from folding on itself. The samples
are separated according to their size on this gel. Then the separated RNA samples are stained
with Ethidium bromide to visualize the RNA bands.

Step 3
Transfer of RNA to nylon membrane:
In this step the agarose gel is blotted on a nitro cellulose filter paper or on a nylon membrane
by creating a sandwich type arrangement. The separated RNA fragments are transferred on this
membrane. Nitrocellulose can binds about 100cm while nylon can binds about 500cm. Many
scientists suggest that nylon is better than nylon and it binds more easily. Nylon is also less
fragile.

Then the RNA is immobilized on the membrane through baking at high temperature 80C or by
UV cross linking which results in covalent linkage of RNA to membrane. It prevents nucleic acid
from being washed away from subsequent processing.

Step 4

Pre hybridize before hybridization:


In the forth step the membrane is placed in a dish that contains hybridization buffer that have
similarity with a labeled probe. If the sample of RNA contains complementary RNA sequence
the probe will bind to the membrane to form double standard DNA-RNA hybrid molecule
between the single standard DNA probe and single standard target RNA. Then it hybridizes to
the RNA on the blot that to the sequence of interest.

Finally the membrane is washed to remove unbound probe.

Step 5

Autoradiography:
in this last step the detection of RNA of interest on the membrane is done by using
autoradiography. The sample is then exposed to X ray film. The fragment containing the gene of
interest can be identified by a band that will develop on the

film.

Application of Northern blotting:


 There are many applications of Northern blotting that are following as:
 Northern blots are very useful in determining the conditions under which specific genes
are expressed.
 This technique is also used to show the over expression of oncogenes and down
regulation of tumour suppressor genes in cancerous cells.
 Northern blotting is used for studying mRNA splicing.
 It is used in detecting a specific mRNA in sample used for screening recombinant which
are transformed with transfer.
 It is also used for studying mRNA splicing.

Conclusion:
This molecular technique is based on PAGE-Northern blot using cDNA. Since this
technique has its vast applications in medical sciences that will create many facilities for
the mankind in future.

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