1. RNA Extraction

2. Ribonucleic acid

3. Inactivating Cellular RNase
Cellular RNases should be inactivated as quickly as possible at the very first stage in the extraction process.
use strong denaturants such as guanidinium hydrochloride or guanidinium thiocyanate to disrupt cells, solubilize their components, and denature endogenous RNases simultaneously

4. Obtaining high quality RNA is the 1st and most important step
Proper handling and use of RNase-free materials will eliminates degradation of RNA and introduction of RNases
Storage of isolated RNA
Store in RNase – free solution
- Store at -70 C
Extraction

5. RNase Contamination
Hands and dust may be the major source of RNase contamination
Use sterile tubes and tips
Use gloves (Powder free)
Store RNA -70°C

6. Lysis step will rupture the cell membrane and release cellular components and nucleic acid from the cell

7. TRIZOL Ready to use reagent for the isolation of total RNA
Solution of phenol and guanidine isothiocyanate

8. Trizol Extraction
- Chloroform is added for phase separation allowing collection of the aqueous phase containing RNA
- RNA is precipitated with addition of Isopropanol
-RNA precipitate is often invisible before centrifugation
- Final wash with ethanol

9. RNA Pellet -Briefly dry pellet for 5-10 min.
-Do not let pellet dry completely or over-dry as this will decrease solubility however
- all residual ETOH must be removed

10. RNX-Plus (CinnaGen) Precipitate cells
Dissolve pellet of cells in 1 ml RNX;
Promotes formation of complexes of RNA with guanidinium and water molecules and abolishes hydrophilic interactions of DNA and proteins.
DNA & proteins are efficiently removed from aqueous phase
RNA remains in aqueous phase

11. RNA extraction by RNX Add 200μl Chloroform Shake (Do not vortex!)
Stay on ice for 15 min
Centrifuge at g
There are 2 phases:
Lower color phenol-chloroform phase
Colorless upper aqueous phase (RNA)
Interphase (DNA & proteins )

12. RNA Precipitation Transfer the aqueous phase to the fresh tube
Add equal vol. of isopropanol
Shake gently
Incubate on ice 30 min
Centrifuge 15 min at 12000g
There will be white pellet at the bottom of the tube.

13. RNA Wash Wash the RNA pellet once wih 1ml 75% Ethanol
Centrifuge 7,500g
Dry pellet
Dissolve RNA pellet in 20-50μl DEPC (Diethylpyrocarbonate) or autoclaved water

14. OD260/280 ratio higher than 1.9
Gel electrophoresis
28srRNA
18srRNA