1. Presented By : *M. UZAIR NIAZ *WAQAR ALI
NORTHERN BLOTTING
Presented By :
*M. UZAIR NIAZ
*WAQAR ALI

2. Contents What is Blotting? Northern Blotting
History and introduction
Principles of Northern Blotting
Procedure
Advantages and disadvantages
Question answers

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 but it analyzes RNA
To study gene expression by detection of RNA during differentiation, diseased conditions

5. Principle of Northern Blotting
Electrophoresis – Seperates 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 Extraction of RNA Electrophoresis of RNA
From homogenised tissue sample
RNA with poly A tail
Electrophoresis of RNA
In agarose gel
RNA is not fragmented
Transferred to nylon paper or DBM paper
Because probes are unable to bind with RNA in gel
Done by capillary action
Buffer contains formamide to reduce annealing temp.

7. Stabilization Hybridization Detection By Heat or UV rays
Formation of covalent linkages
Hybridization
With radiolabelled or fluorescently labelled probe
Detection
By X-rays

8. Diagrammatic View

9. Advantages Disadvantages Simple method Highly specific
Quality and quantity of gene can be measured
Disadvantages
Detect small number of genes at a time
Use of ethidium bromide and UV light needs special training
Detection with multiple probes is difficult

10. THANK YOU 