Thursday, 1 July 2021

Northern Blotting Steps

 Northern Blotting is a blotting technique for the detection of RNA. the overall technique and the steps involved in northern blotting is almost similar to the southern blotting. Northern Blotting is a technique which will be suitable for a study  gene expression and analysis.

Steps of Northern Blotting

1) RNA gel Electrophoresis

• The first step in northern blotting is RNA gel electrophoresis. The RNA molecules isolated from cells are separated according to size by Gel electrophoresis.

• RNA molecules are negatively charged, so they move from negative to the positive electrode during gel electrophoresis.

• RNA is a single-stranded nucleic acid but, still this RNA gel electrophoresis also includes the denaturation step.
- This is because RNA molecules fold onto themselves. Because of intramolecular base pairing they form secondary structures. So if we want to separate them on the basis of their molecular weights we need them to bring in the linear shape.
- Otherwise the secondary structures of RNA molecules will affect their electro phoretic mobility during gel electrophoresis.

• To denature RNA formaldehyde is used as a denaturing agent. Thus denaturring gel electrophoresis is used in this step.


2) Blotting (RNA)

• The second step is blotting.
The separated RNA molecules are now transferred from the gel to the suitable solid support, such as the Nylon membrane.

• There is the traditional way to transfer the RNA to the Nylon membrane. The basis of this transfer is the capillary action.  

System of Northern Blotting

  The tray is filled with the suitable transfer buffer. A support for gel on membrane is kept in this buffer. This is usually a glass plate an absorbent paper or blotting papers are placed on this support. These are placed such that they imbibe the buffer. They act as a wick

-  Next the gel containing RNA is placed on the top of it.
-  An nylon membrane of the same size as that of the gel is placed over it.
- Again a thick stack of blotting papers or absorbent paper is placed over the membrane.
- This is followed by three to four inches of paper towels and this complete stack of gel membrane blotting papers and paper towels are pressed down by putting a weight on top the buffer or liquid.

  From the tray now Rises through the gel taking with it the RNA molecules. Once the RNA molecules reach the nylon membrane they become adsorbed tightly to the membrane.

  The remaining liquid passes through the paper and is absorbed by the paper towels placed at the top. During this transfer the RNA fragments retain the same pattern of separation they had on the gel.  

3) Hybridization and washing

• The third step involves hybridization with probe and washing. Suppose these bands are the RNA molecules on the nylon membrane. For the detection of these RNA molecules first we need a probe that will specifically bind to these target RNA molecules.

• The probe can be a complimentary labeled RNA sequence or labelled complementary DNA sequence.

• When nylon membrane is incubated with these probe molecules probes will bind specifically to their complimentary target RNA molecules.

• Unbound probes are removed by washing.

4). Detection

• In the fourth and final step detection is done. Detection and visualization method depends on the type of labelled molecule we used for hybridization step.

• The probe is detected by autoradiography, fluorescence or a color change depending on what label we have used in the probe.

These were the steps involved in the northern blotting.

Applications of Northern Blotting

The main applications of northern blotting include
• Gene expression studies such as to determine when and where a particular gene is expressed.
• To identify the presence of closely related species
• To determine the size and abundance of RNA.
• For the analysis of RNA processing.

Modern methods such as PCR have replaced the methods of southern and northern blotting. This is because PCR and PCR based techniques are more simple quick and of more precise nature.

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