NORTHERN BLOTTING

By –AKSHAY PAREEKM.Sc. Biotech II sem

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

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.

ProcedureElectrophoresis of RNA

In agarose gelRNA is not fragmented

Transferred to nylon paper or DBM paper• B

ecause probes are unable to bind with RNA in gel

• Done by capillary action

• Buffer contains formamide to reduce annealing temp.

Stabilization

By Heat or UV raysFormation of covalent linkages

Hybridization

With radiolabelled or fluorescently labelled probe

Detection• B

y X-rays

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 bromide and UV light needs

special training• Detection with multiple probes is difficult

THANK YOU