rna extraction. ribonucleic acid inactivating cellular rnase cellular rnases should be inactivated...

RNA Extraction

Ribonucleic acid

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

Extraction 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

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

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

TRIZOL

• Ready to use reagent for the isolation of total RNA

• Solution of phenol and guanidine isothiocyanate

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

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

RNX-Plus (CinnaGen)

Precipitate cellsDissolve 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

RNA extraction by RNX

• Add 200μl Chloroform • Shake (Do not vortex!)• Stay on ice for 15 min• Centrifuge at 12000 g There are 2 phases:Lower color phenol-chloroform phaseColorless upper aqueous phase (RNA)Interphase (DNA & proteins )

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.

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

• OD260/280 ratio higher than 1.9• Gel electrophoresis

28srRNA

18srRNA