Isolation of RNA
- 1. Isolation of RNA Yashswee Ghorpade. Assistant professor Dr. D. Y. Patil ACS College Pimpri Pune.
- 3. 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. Requirements •Potassium acetate •Sodium acetate •Ice cold-ethyl alcohol •Sodium Dodecylsulphate •Extraction buffer: Tris-CL, sodium chloride. Sodium EDTA •Suspension buffer : Tris-HCL , Sodium EDTA •Tris EDTA buffer (TE buffer): Tri-HCL, Sodium EDTA
- 5. Take plant material (bean seed/ cauliflower florets)and cut into small pieces. Add the extraction buffer and make the fine paste Transfer the homogenate to flask add SDS and mix it then incubate at 650C for 15 min in water bath Add potassium acetate solution mix it and incubate for 30min Centrifuge the content at 20000rpm for 15 min. Take supernatant in tubes and add sodium acetate and 95% of ethanol. place the tube in ice bath for 15min for precipitate RNA Then centrifuge the tube on 20000rpm for 15min and discard supernatant And pellet wash with ice cold ethanol and dry it.
- 6. Isolation and preparation of Cytoplasmic RNA from tissue culture cells Requirement : Lysis buffer Dithiothretiol, guanidine isothicyanate, 0.5% N-lauryl sarcosine, sodium acetate. Phosphate buffered saline, SDS, proteinase K, phenol-chloroform- isoamyl alcohol(25:24:1), chloroform-isoamyl alcohol(24:1), sodium acetate, ansolute ethanol, 70% ethanol. TRIzol Reagent
- 7. Take cell suspension culture in centrifuge tube and centrifuge at 3000rpm for 5 min. Re suspend collected cells in PBS , centrifuge it and discard supernatant Re-suspended cell in lysis buffer and incubate on ice bath for 5 min. Centrifuge tube for 2min at 40C and transfer supernatant in new tube and add SDS then vortex it. Add proteinase K and incubate at 370C for 15min. Add phenol-chloroform-isoamyl alcohol and vortex it for 1min and centrifuge the tube. Transfer upper layer in new tube and repeat above step 2 times. New supernatant add sodium acetate and ethanol ,mix it well and incubate on ice bath for 15-30 min for precipitate.
- 8. Isolation of total and polymer RNA Requirements : • RNA extraction buffer: Lithium chloride, LiCi, SDS, Tris- NaOH, EDTA •Lithium chloride( LiCi) 8M: dissolve 34 g of LiCi in 100ml of water and autoclave store in freeze. •Distilled phenol with 0.1% hydroxyquinoline , chloroform , ethanol . •Diethylpyrocarbonate water (DEPC)
- 9. • 1:1mixture of RNA extraction buffer and phenol with hydroxyquinoline and heat it to 90oCin water bath. • take plant material and add liquid nitrogen and grind it fine homogenous powder. • Homogenate to clean flask on ice bath and phenol extraction buffer and gently mix. • Transfer flask in water bath ,incubate sample at 90oC then it forms milky suspension. • Suspension cool on room temp. and centrifuge it at 300rpm for 5min • Then add chloroform and incubate shaking incubator for 30min. •Centrifuge these suspension at 20000rpm for 30 min . •Remove aqueous phase and transfer it into new flask add chloroform. •Shake the content at 3000rpmfor 15 min.centrifuge content at 12000rpm for 15 min. •Remove upper phase and transfer it into new centrifuge tube add LiCi ,mix it and precipitate the RNA for 16-48 hr at 4oc •Centrifuge precipitate at 4oC on 12000rpm for 30min and wash pellet with LiCi and twice with ethanol. •Dry pellet and suspended it in DEPC and store in freeze
- 10. Extraction of polysomes and polysomal RNA Material : Polysome buffer : Nonidet P40, MgCl, EGTA, Tris-NaOH, NaCl. Gradient buffer : MgCl2, EGTA, Tris-NaOH, NACl. GB+ EDTA:75% W/V sucrose in GB: sucrose, DEP RNA extraction buffer: LiCl, phenol, chloroform, ethanol.
- 11. Harvest the plant material and grind in liquid Nitrogen Transfer powder add plant material in ice-cold polysome buffer and form the slurry of these material This mixture/slurry cenrifuge at 27000rpm for 10min at 00C. Filter the supernatant solution. Filtrate into Beckmann polycarbonate tube with sucrose in GB . Centrifuge this for 3hr at 40000rpm then place the tube inverted on sterile paper towel. dry pellet re-suspended in frozen liquid nitrogen and store at -800C Carefully load upto 5 mg polysome on gradient and centrifuge for 30min at 40000rpm Polysome containing fraction are collected and precipitate overnight in 96% ethanol. These precipitate centrifuge at 20000rpm for 30min Extraction of RNA from ethanol precipitate polysome by phenol/chlorofrom/ LiCl method.
- 12. Isolation of bacterial RNA Requirements : 1. STET lysing solution 2. Baffered phenol 3. Chlorofrom 4. Sodium acetate 5. Vanadyl robonucleoside (VRC) 6. Buffered phenol:chlorofrom(1:1) 7. Cesium chloride 8. EDTA 9. Ethanol
- 13. Collect the bacterial culture at log phage and centrifuge it at 5000rpm for 5 min. Resuspend the pellet in lysis buffer and add VRC Add buffered phenol, vortex it for 1 min. add chloroform and vortex it 1 min. Centrifuge the content for 10 min at 10000rpm and collect aqueous phase in new tube Add sodium acetate and 100% ethanol mix it well and centrifuge it for 10 min at 10000rpm then re suspend pellet in VRC Extract pellet with phenol:chlorofrom and re precipitate the RNA same as above step and dry the pellet and dissolve it in sterile D/W and store at -70oC