Northern blotting ppt

Steps in northern blotting

1. ABT 601- PRINCIPLES OF BIOTECHNOLOGY (2+1) NORTHERN BLOTTING 1 LATHA.M 2017601306 NANOSCIENCE &TECHNOLOGY

2. Northern Blot - RNA Developed by James alwine, David Kemp and George Stark – 1977 . Detects the presence of specific mRNA in total RNA extract. Can determine whether the gene is transcribed or not. RNA is more susceptible to degradation than DNA. 2

3. Introduction Concept: To study the gene expression by detection of RNA by molecular biology using Northern blotting. Reannealing nucleic acids to identify sequence of interest. Separates RNA in an agarose gel, then detects specific bands using probe and hybridization. Hybridization takes advantage of the ability of a single stranded DNA or RNA molecule to find its complement, even in the presence of large amounts of unrelated DNA. Allows detection of specific bands (RNA molecules) that have complementary sequence to the probe. Size bands and quantify abundance of molecule. 3

4. Northern blotting Steps 1. Isolate RNA & treat with formaldehyde. 2. Electrophorese RNA in denaturing agarose gel (has formaldehyde). Visualize RNA in gel using Ethidium bromide stain and photograph. 3. Transfer single-stranded RNA to nitrocellulose or nylon membrane. Covalently link RNA to membrane. 4. Incubate membrane (RNA immobilized on membrane) with labeled DNA or RNA probe with target sequence. 5. Autoradiography. 4

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6. Step 1 Isolate RNA: - To detect rare mRNA, isolate the poly A + mRNA. -RNA is both biologically and chemically more labile than DNA. Thus eliminate RNases. 6 Electrophoresis: - Performed in formaldehyde agarose gel to prevent RNA from folding on itself. -Stain with EtBr to visualize the RNA bands. Step 2

7. Step 3 Transfer single-stranded RNA to nitrocellulose or nylon membrane : Traditionally, 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. Covalently link RNA to membrane : UV cross linking is more effective in binding RNA to the membrane than baking at 80  C. 7

8. Step 4 & 5 Prehybridize 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 : Probe could be 32 P, biotin/streptavidin or a bioluminescent probe. Autoradiography : Place membrane over X-ray film. X-ray film darkens where the fragments are complementary to the radioactive probes. 8

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10. Ethidium bromide is fluorescent in UV light 10

11. Agarose gel & Autoradiography 11

12. Northern blotting 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. Detection of specific mRNA in a sample. Used in screening of recombinants by detecting the mRNA produced by the transgene. In disease diagnosis. In gene expression studies. RNA splicing studies. 12

13. Advantages : The strength of this method is its simplicity. Specificity is relatively high. The quality and quantity of RNA can be measured on the gel prior to blotting. mRNA transcript size can be detected. Disadvantages : Time consuming procedure. RNA samples can be degraded by RNases. Use of radio active probes. 13

14. REFERENCES: https:// en.wikipedia.org/wiki/Northern_blot www.biologyexams4u.com/2014/01/northern-blotting-principle-summary-of.html https://en.wikibooks.org/wiki/ Structural_Biochemistry /DNA... techniques/ Northern_Blot https://www.biotecharticles.com/...products.../Technique-of-Northern-Blotting-382.ht ... https://www.youtube.com/watch?v=KfHZFyADnNg 14

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