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Northern blotting

Northern blotting is a technique developed in 1977 to detect specific RNA sequences through electrophoresis, transfer to a membrane, and hybridization with a probe. RNA is extracted from tissue samples, separated by size via electrophoresis in an agarose gel, and transferred by capillary action to a nylon or nitrocellulose membrane where it binds. Probes are then hybridized to the RNA and detected through radiolabeling or fluorescence to study gene expression under different conditions.

• 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
• 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
Northern blotting
Northern blotting
Northern blotting