Northern-Blotting

Northern Blotting: Principle, Steps involved, Advantages and Uses & Disadvantages

Northern Blotting

A northern blot is a laboratory technique used to identify particular RNA molecules among a mixture of RNA. Northern blotting can be used to analyze a sample of RNA from a particular tissue or cell key in order to determine the RNA expression of particular genes.

Northern blotting was the first procedure developed for evaluating the molecular size and abundance of selective RNAs in a mixture of RNAs or nucleic acids.

This process trusts the process of nucleic acid hybridization in between a recognized nucleic acid probe and the complementary sequence in a population of RNAs.

The northern blot method was developed in 1977 by James Alwin, David Kemp, and George Stark at Stanford University, with contributions from Gerhard Heinrich. Northern blotting takes its name from its similarity to the first blotting technique, the Southern blot.

Principle of Northern blotting

Northern blot first uses denaturing gel to separate RNA according to the size. The RNA is then transferred to a nylon membrane while keeping the exact same distribution in the gel. After fixing the RNA to the membrane, a labeled probe complementary to the gene of interest is then added to hybridize to the debilitated RNA.

The nonspecifically bounded probes are then washed away. The solid membrane with probe specifically bound to RNA of interest is then dried, exposed, and evaluated.

Given that northern blot uses size-dependent separation, this strategy can not only figure out the abundance but likewise the sizes of a transcript of interest.

Equipment required
  • Agarose gel rig
  • Power supply
  • Vacuum gel transfer system
  • Nylon membrane
  • Phosphor screen
  • Phosphor screen scanning equipment
  • Hybridization oven
  • Hybridization bottles
Steps involved in Northern blotting

Steps-involved

RNA is isolated from several samples such as skin, hair, the cornea of eyes, sperm/ egg cells.

Samples are loaded in agarose gel.

The RNA sequence is separated in the electrophoresis system an agarose gel is used for the function of the nucleic acid separation.

Now the separated RNA sequence is transferred to the nylon membrane. This is done by two systems capillary action and ionic interaction.

The transfer is done by keeping the gel in the sandwich arrangement. First, the agarose gel is placed on the bottom of the stack, followed by the blotting membrane. On top of these paper towels, a mild weight (glass plate) is positioned. The entire setup is kept in a beaker including a transfer buffer.

RNA transferred to the nylon membrane is then fixed using UV radiation.

The fixed nylon membrane is then combined with probes. The probes are specifically developed for the gene of interest so that they will hybridize with RNA series on the blot corresponding to the sequence of interest.

The blot membrane is washed to get rid of unwanted probes.

The labeled probe is detected by chemiluminescence or autoradiography. The outcome will be dark bands in x-ray film.

Advantages and Uses
  • Northern blotting can be utilized to determine novel splice versions, pre-processed RNAs, and non-coding RNAs, along with their relative abundances.
  • This technique reveals the identity, number, activity, and size of the particular gene.
  • This blotting technique can also be utilized for the development of a tissue or organism.
  • In different phases of differentiation and morphogenesis, the abundance of RNA modifications and can be identified using this strategy.
  • It also helps in the recognition of unusual, diseased, or infected conditions at the molecular level.
Disadvantages
  • Northern blotting is relatively less sensitive than RTPCR.
  • Detection of multiple probes is a problem.
  • Non-specific matching of the probe to RNA can take place.
  • RNA is slightly degraded by RNase due to contamination by water, contaminated hands, or tips and equipment can negatively affect the quantitation.