NORTHERN BLOTTING

PRESENTER:POURAKH GURUNG

PRAMOD RAI

INTRODUCTION Northern blotting was developed by

Alwine in 1977. It a laboratory technique used to study gene expression by detection of RNA in a sample.

Northern blotting is based on the Hybridization principle

MAJOR STEPS INVOLVED RNA isolation Separation of RNA using Gel

Electrophoresis Blotting Hybridization and Washing of excess

probes visualization

RNA ISOLATION The RNA is isolated from the cell.

SEPARATION OF RNA Formaldehyde agarose gel

electrophoresis is normally used in the separation of RNA as formaldehyde agarose gel prevent RNA from folding on itself.

On electrophoresis RNA molecules moves towards positive pole as RNA is negatively charged.

BLOTTING Simply blotting is process of transferring the RNA

molecules to the nitrocellulose membrane or nylon membrane:

Nitrocellulose typically has a binding capacity of about 100µg/cm, while nylon has a binding capacity of about 500 µg/cm. Many scientists feel nylon is better since it binds more and is less fragile.

Once the RNA has been transferred to the membrane, it is immobilized through covalent linkage to the membrane by UV light or heat.

UV cross linking is more effective in binding RNA to the membrane.

Fig. Capillary blotting system setup for the transfer of RNA from an electrophoresis gel to a blotting membrane.

HYBRIDIZATON AND WASHING OF EXCESS PROBS

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.

The probes must be labelled either with radioactive isotopes (32P) or with chemiluminescence.

Hybridization between probes and the target RNA Washing of excess probes

VISUALIZATIONAutoradiography:

Place membrane over X-ray film.X-ray film darkens where the fragments are complementary to the radioactive probes.

SUMMARY

NORTHEN BLOT APPLICATION Northern blots are particularly useful for

determining the conditions under which specific genes are being expressed, including which tissues in a complex organism express which of its genes at the mRNA level.

For instance: When trying to learn about the function of a certain protein, it is sometimes useful to purify mRNA from many different tissues or cell types and then prepare a Northern blot of those mRNAs, using a cDNA clone of the protein of interest as the probe. 

Only mRNA from the cell types that are synthesizing the protein will hybridize to the probe.

Northern blotting allows one to observe a particular gene's expression pattern between tissues, organs, developmental stages, pathogen infection, and over the course of treatment

 The technique has been used to show overexpression of oncogenes and downregulation of tumor-suppressor genes in cancerous cells when compared to 'normal' tissue, as well as the gene expression in the rejection of transplanted organ.

references www.weikepedia.com http://homepage.smc.edu/colavito_mary/biology22/2 www.colorado.edu/MCDB/MCDB2150Fall/notes99/99L17.html www.uwp.edu/~higgs/Lect12mb.html

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