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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
• 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

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Northern blotting

  • 2. • 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
  • 3. • 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.
  • 4. 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.
  • 5. Stabilization • By Heat or UV rays • Formation of covalent linkages Hybridization • With radiolabelled or fluorescently labelled probe Detection • By X-rays