VPB 321: Animal Biotechnology (2+1)

STEPS IN NORTHERN BLOTTING

• RNA isolation
  • The total RNA is extracted from homogenized tissue samples. The mRNA is isolated by using an oligo-dT column
• Gel electrophoresis of RNA for separation
  • The mRNA is separated on an agarose gel containing formaldehyde as a denaturing agent for the RNA to limit secondary structure.
• Transfer to membrane (usually positively charged nylon as RNA is negatively charged)
• Cross-linking of RNA to membrane (usually by UV-crosslinking or chemical means)
• Hybridization
  • Probes for northern blotting are composed of nucleic acids with a complementary sequence to all or part of the RNA of interest, they can be DNA, RNA, or oligonucleotides with a minimum of 25 complementary bases to the target sequence.
  • Commonly cDNA is created with labelled primers for the RNA sequence of interest to act as the probe in the northern blot.
• Detection
  • The probes need to be labelled either with radioactive isotopes (³²P) or with chemiluminescence.  X-ray film can detect both the radioactive and chemiluminescent signals and many researchers prefer the chemiluminescent signals because they are faster, more sensitive, and reduce the health hazards that go along with radioactive labels.
  • The same membrane can be probed up to five times without a significant loss of the target RNA.