Presented By : *M. UZAIR NIAZ *WAQAR ALI 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 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.

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

Diagrammatic View

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

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