northern blotting

NORTHERN BLOTTING

PRESENTED BY :

*M. UZAIR NIAZ*WAQAR ALI

Contents

What is Blotting? Northern Blotting

History and introduction Principles of Northern Blotting Procedure Advantages and disadvantages Question answers

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.

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.

Procedure

Extraction 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.

Stabilization• By Heat or UV rays• Formation of covalent linkages

Hybridization• With radiolabelled or fluorescently labelled

probe

Detection• By X-rays

Diagrammatic View

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

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