northern blotting

1. Seminar Presentation on
Northern Blotting
Presenting By:
Mohsin Ali
Student of MSc Medical Virology 1st Year
Jamia Hamdard
New Delhi

2. Overview
What is Blotting?
Northern Blotting
History and Introduction
Principles of Northern Blotting
Procedure
Advantages and Disadvantages
Applications of Northern Blot

3. Blotting
Blotting is the technique in which nucleic acids or
proteins are immobilized onto a solid support generally
nylon or nitrocellulose membranes.
Blotting of nucleic acid is the central technique for
hybridization studies.
Nucleic acid labeling and hybridization on membranes
have formed the basis for a range of experimental
techniques involving understanding of gene
expression,organization,etc.

4. Northern Blotting
Developed by James Alwine,David Kemp and George
Stark in 1977.
Similarity of name with Southern Blotting it analyzes
RNA
To study gene expression by detection of RNA during
differentiation,Diseased,Conditions

5. Principle of Northern Blotting
Electrophoresis- Separates RNA on the basis of their
Molecular weight and type in agarose gel which have
EtBr, an intercalating agent in it.
Capillary action- RNA bands move towards blotting
paper by capillary movement and entrap in sheet and
buffer moves ahead.

6. Procedure for Northern Blotting
1. Tissue or culture sample collected (Homogenised)
2.RNA sequence is separated in the electrophoresis
unit (Agarose Gel)
3. Separated RNA molecule is transferred to the Nylon
Membrane (Capillary action and Ionic Interaction)
4.Agarose gel is place on the bottom of the stack,
follow by blotting membrane.
5.Entire setup is kept in a beaker containing transfer
buffer.

7. 6.RNA transferred to the nylon membrane is then
fixed with UV radiation.
7. The fixed nylon membrane is then mixed with
probe (specially designed for the gene of interest)
8.The blot membrane is washed to remove unwanted
probe
9.Labled probe is detected by chemiluminescene or
autoradiography.The result with be dark bands in
X-ray film

8.  Procedure

9. Diagrammatic View

10. Advantages
Simple Method
Highly Specific
Quality and Quantity of gene can be measured
Disadvantages
Detect small number of genes at a time
Use of ethidium and UV light needs special training
Detection with multiple probe is difficult

11. Applications of Northern Blot
• The technique can be used for the identification and separation of RNA fragments collected from
different biological sources.
• Northern blotting is used as a sensitive test for the detection of transcription of DNA fragments that
are to be used as a probe in Southern Blotting.
• It also allows the detection and quantification of specific mRNAs from different tissues and different
living organisms.
• Northern blotting is used as a tool for gene expression studies related to overexpression of cancer-
causing genes, and gene expression during transplant rejects.
• Northern blotting has been used as a molecular tool for the diagnosis of diseases like Crohn’s disease.
• The process is used as a method for the detection of viral microRNAs that play important roles in viral
infection.

12. References
https://www.youtube.com/watch?v=11NHYntRV4Q&t=310s
https://pubmed.ncbi.nlm.nih.gov/18428227/