Northern Blotting Presentation.pptx

Northern Blotting Presentation.pptx

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  Seminar Presentation on NorthernBlotting Presenting By: Mohsin Ali Student of MSc Medical Virology 1st Year Jamia Hamdard New Delhi
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  Overview What is Blotting? NorthernBlotting History and Introduction Principles of Northern Blotting Procedure Advantages and Disadvantages Applications of Northern Blot
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  Blotting Blotting is thetechnique in which nucleic acids or proteins are immobilized onto a solid support generally nylon or nitrocellulose membranes. Blotting of nucleic acid is the central technique for hybridization studies. Nucleic acid labeling and hybridization on membranes have formed the basis for a range of experimental techniques involving understanding of gene expression,organization,etc.
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  Northern Blotting Developed byJames Alwine,David Kemp and George Stark in 1977. Similarity of name with Southern Blotting it analyzes RNA To study gene expression by detection of RNA during differentiation,Diseased,Conditions
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  Principle of NorthernBlotting Electrophoresis- Separates RNA on the basis of their Molecular weight and type in agarose gel which have EtBr, an intercalating agent in it. Capillary action- RNA bands move towards blotting paper by capillary movement and entrap in sheet and buffer moves ahead.
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  Procedure for NorthernBlotting 1. Tissue or culture sample collected (Homogenised) 2.RNA sequence is separated in the electrophoresis unit (Agarose Gel) 3. Separated RNA molecule is transferred to the Nylon Membrane (Capillary action and Ionic Interaction) 4.Agarose gel is place on the bottom of the stack, follow by blotting membrane. 5.Entire setup is kept in a beaker containing transfer buffer.
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  6.RNA transferred tothe nylon membrane is then fixed with UV radiation. 7. The fixed nylon membrane is then mixed with probe (specially designed for the gene of interest) 8.The blot membrane is washed to remove unwanted probe 9.Labled probe is detected by chemiluminescene or autoradiography.The result with be dark bands in X-ray film
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   Procedure
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  Diagrammatic View
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  Advantages Simple Method Highly Specific Qualityand Quantity of gene can be measured Disadvantages Detect small number of genes at a time Use of ethidium and UV light needs special training Detection with multiple probe is difficult
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  Applications of NorthernBlot • The technique can be used for the identification and separation of RNA fragments collected from different biological sources. • Northern blotting is used as a sensitive test for the detection of transcription of DNA fragments that are to be used as a probe in Southern Blotting. • It also allows the detection and quantification of specific mRNAs from different tissues and different living organisms. • Northern blotting is used as a tool for gene expression studies related to overexpression of cancer- causing genes, and gene expression during transplant rejects. • Northern blotting has been used as a molecular tool for the diagnosis of diseases like Crohn’s disease. • The process is used as a method for the detection of viral microRNAs that play important roles in viral infection.
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  References https://www.youtube.com/watch?v=11NHYntRV4Q&t=310s https://pubmed.ncbi.nlm.nih.gov/18428227/