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Rna isolatn

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  3. 3. What is RNA? • Ribonucleic Acid (RNA) is a polymeric molecule. • It is implicated in various biological roles in coding, decoding, regulation, and expression of genes • Found in the nucleus and the cytoplasm • RNA (ribonucleic acid) store and transfer genetic information in living organisms. • Three major type of RNA, mRNA , tRNA and rRNA 3
  4. 4. INTRODUCTION  Ribonucleic acid can be isolated from plant tissue for the purpose of: – mRNA isolation – In vitro translation – Northern analysis – cDNA library construction  Rigorous ribonuclease free environment is to be maintained  All glasswares, plasticwares and reagents made RNAse free (using 0.01% DEPC)  Next day, DEPC is inactivated by autoclaving for 30 min 4
  6. 6. 6 • 4M Guanidine thiocyanate, 42 mM sodium citrate, 0.83% N- lauryl sarcosine and 0.2 mM 2- mercaptoethanol Denaturing buffer • Saturated with 50mM sodium acetate till the pH of upper aqueous phase is approximately 4.0 Phenol • Acid saturated phenol: chloroform: isoamyl alcohol(25:24:1) Phenol- chloroform CONTINUE………
  7. 7. 7 Collect, wash, blot dry and weigh fresh tissues Grind tissue to fine powder under liquid nitrogen and extract with 4ml/gm pre-chilled denaturing buffer Transfer the homogenate to centrifuge tubes Add sodium acetate and centrifuge the slurry for 20 minutes Transfer supernatant to other tube and add phenol chloroform PROCEDURE Mix thoroughly and chill on ice for 15 minutes
  8. 8. 8 Separate the phases by centrifugation Remove the top aqueous phase containing RNA carefully to fresh tubes Add isopropanol and allow RNA to precipitate overnight Next day, RNA is pelleted by centrifugation at 10,000 rpm Resuspend the RNA pellet and heat briefly upto 65⁰C Reprecipitate and pellet RNA with isopropanol Resuspend the pellet in 75% ethanol and centrifuge it Pellet is dried briefly under vacuum , resuspend in RNAse free water and stored at -70⁰C
  9. 9. PRECAUTION A separate corner should be in lab for RNA work which is kept free from RNAse contaminant Phenol have to be acid saturated to a pH nearing 4.0 High concentration of Guanidine thiocyanate should be used Before removing Guanidine thiocyanate, all the protein should be removed Disposable gloves should be used and changed frequently 9
  10. 10. Extraction of RNA CTAB Method • Lysis the cell and all the organelles, to free nucleic acid • Collect the intact mass of nucleic acid by shaking (vortex or centrifuge) • DNase used to eliminate DNA and separate RNA • Before use add 1% beta marcaptoethanol. 10
  11. 11. Materials • Before use add 1% beta marcaptoethanol. 11
  12. 12. PROCEDURE • For tissue lysis, plant materials or fungal spores, hyphae are broken down into small parts by grinding in pestle and mortar adding liquid Nitrogen. • Then 1% CTAB is added to break down tissue. Then it is incubated at 55-65 °C for 30 minutes to overnight. • The incubation is given by increasing temperature. The eppendorf is taken and added 1% CTAB buffer half and tissue material half. • Then it is centrifuged at maximum speed for 10-15 minutes. • All the cell debris or impurities settled down at the bottom after centrifugation and upper liquid is formed which is called supernatant. • Supernatant is shifted to another test tube and mixed with equal amount of chloroform : Isoamyl alcohol. • Then sample is vortex for 3-5 minutes then centrifuged at maximum speed for 10 minutes. 12
  13. 13. • The pellet settles down at bottom and supernatant is shifted to another Eppendorf. • Added equal amount of Iso-propanol and incubated at -20 °C for 5 minutes. • Then is again centrifuged at maximum speed RNA settle down at the bottom. • Then it is washed with 70% ethanol and centrifuged again pellet down at the bottom. • Transfer the liquid into a new eppendorf. • Add LiCl and mix well. This will precipitate the RNA only. • Precipitate the RNA for at least 20 min at -20 °C. 13
  14. 14. • Spin down at max speed for 30 min at 4 °C • Keep the pellet, discard supernatant. The pure RNA pellet might be transparent and hardly visible. • Add 70% ethanol and mix well to wash of the salts. • Spin down at max speed for 2 min • Keep the pellet, discard supernatant • Dissolve pellet in water for 15 min at 65 °C. • Transfer the liquid into a new eppendorf. • Store RNA at -04 °C for a week -20 °C for a month -80 °C for a year 14
  15. 15. TRIzol Method • This is a modification of the procedure originally described by Chomczynski P. and Sacchi N. 1987. • The correct name of the method is guanidinium thiocyanatephenol- 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. 15
  16. 16. Material and Reagents • 1) TRIzol Reagent (Commercially available from many venders) • Phenol in saturated buffer 380 mL 38% • Guanidine thiocyanate 118.16 g, 0.8 M • Ammonium thiocyanate 76.12 g, 0.4 M • Sodium acetate pH 5.0 33.4 mL of 3 M stock 0.1 M • Glycerol 50 mL 5% 16
  17. 17. • 0.8 M sodium citrate / 1.2 M NaClO • Isopropanol (2-Propanol) • Chloroform • DEPC-Water • 75% ethanol prepared with DEPC-Water • RNase Inhibitor (e.g., aseERASE TM BIO 101 Cat. 2601-104) • 50 mL sterile plastic screw-cap centrifuge tubes 17
  18. 18. PROCEDURE • Grind 1g tissue in liquid nitrogen in a mortar and pestle. • Transfer powdered tissue to a sterile plastic screw-cap centrifuge tube containing TRIzol reagent. • Incubate samples at room temperature for 5 min. • Homogenize tissue with homogenizer for 15 seconds. • Centrifuge samples at 12000 rpm at 4°C for 10 min. • Transfer supernatant into new sterile plastic screw-cap centrifuge tube, Discard pellet. • Add chloroform tube and shake it vigorously with vortex for 15 sec. – Let tube placed at room temp 2-3 min. Centrifuge tube at 10,000 rpm at 4°C for 15 min. – Carefully pippet aqueous phase into a clean screw-cap centrifuge tube, discard interphase and lower phase into waste. 18
  19. 19. • Precipitate RNA by adding isopropanol and sodium citrate half volume of the aqueous phase. Cover tube and mix by gentle inversion. Let sit at room temperature for 10 min. • Centrifuge tubes at 10,000 rpm at 4°C for 10 min. Discard supernatant. • Wash pellet with 20 ml of 75% ethanol. Vortex briefly. • Centrifuge at 10,000 rpm at 4°C for 10 min. Discard supernatant and dry pellet. • Add DEPC-Water, to pellet. Resuspend RNA by pipetting up and down a few times. • Add RNase inhibitor aseERASE to RNA sample • Transfer sample to microcentrifuge tube at room temperature. • Spin samples at high speed in microcentrifuge tube for 5 min at room temperature. • Transfer RNA solution (supernatant) to a new tube. And store it. 19
  20. 20. 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. 20
  21. 21. Benefits of organic extraction • Rapid denaturation of nucleases and stabilization of RNA Drawbacks of organic extraction • Laborious and manually intensive processing • Difficult method. 21
  22. 22. 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. 22
  23. 23. Filter-based RNA isolation 23
  24. 24. 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 24
  25. 25. 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. 25
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