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Northern blotting ppt

Northern blotting is a technique used to detect specific mRNAs in a sample. It involves separating RNA fragments by electrophoresis, transferring them to a membrane, then using a probe with a complementary nucleotide sequence to identify particular mRNA transcripts through hybridization. This allows researchers to determine which genes are actively being expressed and study gene expression under different conditions. The key steps are isolating RNA, separating fragments by size in a gel, immobilizing them on a membrane, then incubating with a labeled probe and detecting hybridized bands through autoradiography or other methods. Northern blotting provides a simple way to analyze mRNA levels and splicing variations.

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1 LATHA.M 2017601306 NANOSCIENCE &TECHNOLOGY
 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
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
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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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
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
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 32P, 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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 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
 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
 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/D NA...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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