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RNA isolation

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DIFFERENT METHODS OF RNA ISOLATION

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RNA isolation

  1. 1. RNA ISOLATION ZARA AHMAD KHAN
  2. 2. RNA extraction  RNA extraction is the purification of RNA from biological samples. This procedure is complicated by the presence of ribonuclease enzymes in cells and tissues.
  3. 3. ISOLATION OF RNA BY YEAST  MATERIAL  BUFFER A  • Buffer A saturated phenol (1.2 ml per sample)  • phenol: Chloroform (0.6 ml per sample)  • 3 M NaOAc (pH 5.2) (90 µl per sample)  • DEPC-treated dH2O (1.5 ml per sample)  • Absolute ethanol (2 ml per sample)  • 70 % ethanol (1 ml per sample) •BUFFER A •16.7 ml 3 M NaOAc • 20 ml 0.5 M EDTA •963.3 ml dH2O
  4. 4. PROTOCOL  A. HARVEST CELL
  5. 5. RNA EXTRACTION FROM YEAST
  6. 6. RNA extraction:  1. Remove the tubes from the -70°C and immediately add 500 µl of Complete Buffer A .Vortex to resuspend the cells.  Centrifuge the tubes in a microcentrifuge for 30 sec at full speed.  Remove the layer using an RNase free blue tip.  Add 600 µl of Buffer A again.  Centrifuge the tubes in a microcentrifuge for 2-3 minutes at full speed.  Remove aqueous layer (top layer) to a new tube.  Add 600 µl of 1:1 phenol buffered with chloroform at room temperature. Mix the samples by vortexing for 20 seconds. Separate the layers by centrifuging the tubes in a microcentrifuge for 2-3 minutes at full speed.
  7. 7. EXTRACTION OF RNA  Remove the aqueous layer (top layer) to a new tube. Add 50 µl of 3 M NaOAc (Ph 5.2) and 1 ml of absolute ethanol.  Resuspend the pellets in 400 µl of dH2O  Wash the pellets by adding 1 ml of 70% ethanol and vortexing for 20 seconds.  Centrifuge the microcentrifuge tubes at full speed for 5 minutes.  Remove the supernatant. Incubate the open tube at 37°C for 5 minutes to dry the pellet.  Dissolve the RNA in 50 µl dH2O. Vortex it. Centrifuge briefly.  Dilute 5 µl RNA into 495 µl of dH2O. Determine the  Dilute the RNA to 1 µg/µl
  8. 8. EXTRACTION OF RNA BY YEAST
  9. 9. TRIZOL RNA Isolation Protocol  TRIZOLE REAGENT  The correct name of the method is guanidinium thiocyanate- phenol-chloroform extraction.  TRIzol is light sensitive and is often stored in a dark-colored, glass container covered in foil. It must be kept below room temperature.  When used, it resembles cough syrup, bright pink. The smell of the phenol is extremely strong..  Caution should be taken while using TRIzol (due to the phenol and chloroform).  Exposure to TRIzol can be a serious health hazard. Exposure can lead to serious chemical burns and permanent scarring  . A lab coat, gloves and a plastic apron are recommended
  10. 10. TRIZOL RNA Isolation Protocol
  11. 11. TRIZOL RNA Isolation  ADD ImL OF TRIZOLE TO SAMPLE AND HOMOGINIZE.  ADD 200uL OF CHOLOROFORM TO THE HOMOGENATE AND VORTEX IT.  CENTRIFUGE (12,000g FOR 15 MIN)  TRANSFER AQUEOUS PHASE TO FRESH TUBE.  PRECIPITATE THE RNA BY MIXING WITH 0.5uL ISOPROPANOL.  CENTRIFUGE FOR 10 MIN AT 12,000g AND REMOVE SUPERNATANT  WASH PALLET WITH 1mL OF 70% ETHANOL BY FLICKING.  CENTRIFUGE AT 7,5OOg FOR 10 MIN.  REMOVE SUPERNATANT AND AIR DRY IT.  DISSOLVE RNA PELLET IN APPROPRIATE VOLUME OF RNASE FREE H2O.
  12. 12. TRIZOL RNA Isolation
  13. 13. MORE TECHNIQUES (RNA ISOLATION)  Organic Extraction Methods  Filter-based RNA isolation  Magnetic Particle Methods  Direct Lysis Methods  RNA extraction in liquid nitrogen
  14. 14. MORE TECHNIQUES (RNA ISOLATION)  Organic Extraction Methods  Organic extraction methods are considered the gold standard for RNA preparation.  During this process, the sample is homogenized in a phenol- containing solution and the sample is then centrifuged.  During centrifugation, the sample separates into three phases: a lower organic phase, a middle phase that contains denatured proteins and DNA, and an upper aqueous phase that contains RNA.  The upper aqueous phase is recovered and RNA is collected by alcohol precipitation.
  15. 15. Organic Extraction Methods
  16. 16. Organic Extraction Methods  Benefits of organic extraction  Rapid denaturation of nucleases and stabilization of RNA  Drawbacks of organic extraction  Laborious and manually intensive processing  Difficult method.
  17. 17. Filter-based RNA isolation  Filter-based, spin basket formats utilize membranes that are seated at the bottom of a small plastic basket.  Samples are lysed in a buffer that contains RNase inhibitors (usually guanidine salts),are bound to the membrane by passing the lysate through the membrane using centrifugal force.  Wash solutions are passed through the membrane and discarded. An appropriate elution solution is applied and the sample is collected into a tube by centrifugation. 
  18. 18. Filter-based RNA isolation
  19. 19. Filter-based RNA isolation  Benefits of spin basket formats  Convenience and ease of use  Ability to isolate RNA and DNA.  Ability to manufacture membranes of various dimensions  Drawbacks of spin basket formats  Propensity to clog with particulate material  Retention of large nucleic acids such as gDNA
  20. 20. Magnetic Particle Methods  Magnetic particle methods utilize small (0.5–1 µm) particles that contain a paramagnetic core.  Paramagnetic particles migrate when exposed to a magnetic field, but retain minimal magnetic memory once the field is removed.  This allows the particles to interact with molecules of interest based on their surface modifications, be collected rapidly using an external magnetic field, and then be resuspended easily once the field is removed.  Samples are lysed in a solution containing RNase inhibitors and allowed to bind to magnetic particles. The magnetic particles and associated cargo are collected by applying a magnetic field.
  21. 21. Magnetic Particle Methods
  22. 22. Magnetic Particle Methods
  23. 23. Magnetic Particle Methods
  24. 24. THANK YOU!!!

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