Northern Blotting: Principle, Procedure and Applications

• The technique was developed by Alwine and his colleagues in 1979.
• The Northern blot is used to detect the presence of a particular mRNA in a sample
• The term “Northern” has no scientific significance just a misnomer.

Principle:
The key to this method is hybridization.
Hybridization: It is the process of forming a double-stranded DNA-RNA hybrid molecule between a single-stranded DNA probe and a single-stranded target RNA.
There are 2 important features of hybridization:

• The reactions are specific-the probes will only bind to targets with a complementary sequence.
• The probe can find one molecule of target in a mixture of millions of related but non-complementary molecules.

Summary of procedure
1. Extract and purify mRNA from cells
2. Separated by gel electrophoresis
3. This gel is immersed in depurination buffer for 5-10 minutes and washed with water
4. Transfer to aminobenzyloxymethyl filter paper. The transfer of RNA from gel to membrane is called blotting
5. After transfer, the membrane is baked at 80⁰C
6. Add labelled DNA probe for hybridization to take place
7. Wash off unbound probe
8. Autoradiograph to detect mRNA –DNA hybrid

 Difference between Northern and Southern blotting (Southern vs Northern blotting)

Applications:

• Detecting a specific mRNA in a sample.
• Used in the screening of recombinants by detecting the mRNA produced by the transgene.
• In disease diagnosis.
• In gene expression studies.