Northern blotting

Northern blotting

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  NORTHERN BLOTTING DSE –Animal Biotechnology B.Sc (Life Science) 3rd Year : Rohit Mondal B.Sc(Life Science) 3rd yr Sri Aurobindo College DSE-Cell and Molecular biology
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  What is ablot ?  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 Bloting :  Southern Blotting: Used For DNA Detection in a sample  Northern Blotting : Used For RNA Detection in a sample  Western Blotting : Used For Proteins in a sample
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  What is aNorthern Blotting ?  The northern blotting is a technique used in molecular biology research to study gene expression by detection of RNA.  The Northern blot, also known as the RNA blot, is one of the blotting techniques used to transfer DNA and RNA onto a carrier for sorting and identification.  The Northern blot is similar to the Southern blot except that RNA instead of DNA is the subject of analysis in this technique.  It is mRNA which is isolated and hybridized in northern blots.
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  Discovery of NorthernBlotting  The northern blot technique was developed in 1977 by James Alwine, David Kemp, and George Stark at Stanford University.  J.C. Alwine, a biologist with a sense of humor, developed a technique analogous to the Southern blot, this time for the identification of a specific RNA within a complex RNA sample using a radio- labelled DNA probe. Alwine couldn’t resist the temptation to call his technique the northern blot in an allusion to Southern’s technique, raising a chuckles in labs everywhere.
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  Schematic view ofNorthern Blot • kk
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  Principle of NorthernBlotting  Northern blotting is a method used to study gene expression by detection of RNA in a sample. Therefore,it is also called RNA Blot.  The sample RNA is isolated from an organism of interest and then electrophoresed on agarose gel which separates the fragments on the basis of their size.  The separated RNA fragments are transferred to a support membrane(nitrocellulose membrane). This can be performed by simple capillary method in presence of a specific buffer.
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   The RNAis then immobilized on membrane eitherby baking at high temperature or UV crooslinking,which results in covalent linkage of RNA to membrane preventing nucleic acid from being washed away from subsequent processing.
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   This isfollowed by hybridisation with a labeled DNA or RNA probe. If the sample contains the complementary RNA sequence, the probe will bind to membrane to form double stranded DNA-RNA hybrid molecule between single stranded DNA probe and single stranded target RNA.  The final step is the detection of RNA of interest on the membrane using chromogen.
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  Requirements of NorthernBlotting  Agarose Gel for process of gel electrophoresis.  Nylon membrane/ Diazo benzyl oxy methyl (DBM) filter paper.  Complementary Radioactive probe for hybridisation.  Formaldehyde(HCHO) for degradation - Carbonal group reacts to form stiff base with amino group of GAC, this prevents normal H-bonding & Hence maintain RNA in denatured State.  X-ray film for identification of RNA.  Note -No need of restriction enzyme for Northern Blotting.
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  Procedure  Step 1:DNA containing the gene of interest is exteacted from human cells and cut into fragments by ristriction enzymes.  Step 2: The fragments are separated According to size by gel electrophoresis. Each band consist of many copies of a particular DNA fragment. Thus bands are invisible but can be visible by straining.  Step 3: The DNA bands are transferred to a nitrocellulose filter by blotting. The solution passes through the gel and filter to the paper towels.
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  • Step 4:This produces a nitrocellulose filter with DNA fragments positioned exactly as on the gel. • Step 5: The filter is exposed to radioactivity labelled probe for a specific gene. The Probe will base- pair (hybridise) wiyh a short sequence present on the gene. • Step 6: The filter is then exposed to X ray Film. The Fragment containing the gene of interest is identified by a band on the developed film.
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  Application of Northern Blotting Observe a particular gene's expression,pattern between tissues,organs, development stages,environment stress levelS etc.  Used to show overexpression of oncogenes and down regulation of tumour suppressor gene's in cancerous cells.  Detecting a specific mRNA in sample used for screening recombinant which are successfully transformed with transfer.  Also used for studying mRNA Splicing.
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  Reverse Northern Blotting The reverse northern blot is a method by which gene expression patterns may be analyzed by comparing isolated RNA molecules from a tester sample to samples in a control cDNA library. It is a variant of the northern blot in which the nucleic acid immobilized on a membrane is a collection of isolated DNA fragments rather than RNA, and the probe is RNA extracted from a tissue and radioactively labelled.
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  Advantages of NorthernBlotting  Northern blots are particularly useful to determine conditions under which specific genes are expressed.  Only mRNA from cell types that are synthesizing protein will hybridize to the probe.  It is also useful in detection of mRNA transcript size.Specifity is relatively high.  RNA splicing is visible because alternatively spliced transcripts can be detected.  Blots can be stored for several years and reprobed if necessary.
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  Disadvantages of Northern BlottingRisk of mRNA degradation during electrophoresis: quality and quantification of expression are negatively affected.  High doses of radioactivity and formaldehyde are a risk for workers and the environment  The sensitivity of northern blotting is relatively low in comparison with that of RT-PCR.  Detection with multiple probes is difficult and also time consuming procedure  Use of ethidium bromide, DEPC and UV light needs special training and attention.
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  Precaution used inNorthern blotting  Remove air bubbles trapped between the gel & the membrane.  Ensure that all buffer components are fully dissolved before using.  Ensure that the electrophoresis tanks are rinsed with distilled water after used.  Control the temperature during hybridization.  Always check for the incorporation of radioactive label before using the probe.  Don’t reuse electrophoresis buffer & radioactive probes.
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