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