Northern blotting technique is used to detect specific sequences of ribonucleic acid (RNA). The protocol for performing northern blotting technique is similar to that of southern blotting technique.

Northern blotting technique was first described by James Alwine and colleagues in 1977 as a molecular biology technique for identifying specific nucleotide sequence of a piece of RNA.

While southern blotting technique detect specific sequences of DNA, northern blotting technique exclusively detect specific sequences of RNA (particularly mRNA).

STEPS FOR NORTHERN BLOTTING

• The isolated RNA is digested with restriction endonuclease.

• Perform agarose gel electrophoresis (as earlier described) on the digested RNA.

• The RNA samples are separated according to their different sizes on agarose gel electrophoresis technique.

• Hybridize separated RNA in a neutralizing buffer by soaking the gel slab in an alkaline solution (e.g. NaOH).

• Transfer the gel slab (carrying the fractionated RNA fragments) onto a nitrocellulose membrane by blotting. Practically, the single-stranded RNA molecules are transferred by capillary action to a stack of nitrocellulose paper or membrane from the gel slab.

• Place a weight on the stack of nitrocellulose membrane in order to hold the blot in place. The nitrocellulose membrane is an absorbent paper; and thus the patterns of the RNA fragments are transferred onto the nitrocellulose paper via capillary action. RNA fragments are bound to the nitrocellulose paper in positions identical to those on the gel slab.

• Place the nitrocellulose membrane in a dish containing hybridization buffer with a radioactively labeled probe (specifically designed for the RNA sequence of interest).

• Wash the membrane with distilled water to remove unbound probe.

• Expose the nitrocellulose membrane to an X-ray film, and photograph it by autoradiography. Expression patterns of the RNA sequence of interest in the different samples are compared and analyzed.

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