Northern Blotting Presented TO: Dr. Dildar Hussain Kalhoro Associate Professor Department OF Veterinary Microbiology Presented & Prepared By Hamid UR Rahman 2K16-VM-46

What is Blot?In molecular biology blots is a technique for transferring DNA , RNA and proteins onto a carrier so they can be separated, and often follows the use of gel electrophoresis. Types OF Bolting:Southern Blotting: Used For DNA Detection in a sampleNorthern Blotting : Used For RNA Detection in a sampleWestern Blotting : Used For Proteins in a sample

Northern Blotting

The northern blot is a technique used in  molecular biology to study  gene expression  by detection of RNA (or isolated mRNA) in a sample.Developed by Alwnie and his colleagues in 1979.This method was named for its similarity to the technique known as a Southern blot.

Northern Blotting

Applications 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.

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.

Procedure STEP 1 : ISOLATAION OF RNA :

All the target RNA’s molecules should be extracted from the sample.Isolate RNA from cells or tissue samples using the TRI reagent or genelute™ kit for mammalian cells or tissues.

Step 2 : Agarose Gel Electrophoresis :In gel electrophoresis the mixture of mRNA molecules separated/denatured to small fragments/molecules according to their size using an electric field.

Agarose Gel ElectrophoresisThe sample should be pushed to electrical field through a gel that containing small pores at a speed that are inversely proportional to the size. As we know that DNA/RNA have negative charge due to the sugar-phosphate backbone will move toward positive end of the field.

Agarose Gel Electrophoresis

Agarose & Buffer : Agarose & buffer are used is a gel to conduct electrical current.Formaldehyde : Formaldehyde is used to unfragment the branched RNA molecule to simple linear one and to prevent it form coiling again.

Gel Electrophoresis

Blotting/ TransferThe transfer or blotting is the step in which the mRNA from the electrophoresis gel will be transferred onto a nylon membrane so it may be accessible to a probe for hybridization and detection. The separated mRNA bands are then blotted on chemically reactive filter paper.

Blotting/ TransferTraditionally, a nitrocellulose membrane is used, although nylon or a positively charged nylon membrane may be used. 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. RNA,s will be covalently link to membrane.

Blotting/ Transfer

Hybridization/ ProbeIn molecular biology hybridization means the process of forming a double stranded nucleic acid from joining two complementary strands of DNA (or RNA).Pre-hybridization before hybridization blocks non-specific sites to prevent the single-stranded probe from binding just anywhere on the membrane.Incubate membrane with labeled DNA or RNA probe with target sequence.

Hybridization/ Probe Incubate for several hours at suitable renaturation temperature that will permit probe to anneal to its target sequence(s).Probe sequence is complementary to the RNA of interest.

Hybridization/ Probe

Washing And Visualization

Wash Nylon from excess of probe & dry.Place Nylon sheet over x-ray film.X-ray film darkens where the fragments are complementary to the radioactive probes.

Advantages/Application

A standard for direct study of the gene expression at the level of mRNA. 

Detection of mRNA transcript size DISADVANTAGE : Time consuming procedure . Use of radioactive probes.  Detection with multiple probes is a problem.

Northern And Southern Blotting Difference

Bothe are same in protocol but some difference as below:Character Southern Northern

Introduction Southern blotting was developed for the very first time by the E.M. Southern in 1975.

This method was developed by the Alwine and his colleagues in 1979.

Function For DNA For RNADenaturation Need of Denaturation No Need Hybridization DNA-DNA

hybridizationRNA-DNA hybridization

Name Southern name of Inventor

Northern is a misnomer