Objective
To Prepare Cytoplasmic RNA from Culture cells
Overview
RNA can be prepared by cell lysis followed by extraction with Phenol/chloroform/Isoamyl and chloroform/alcohol and RNA. The major source of failure in any attempt to
produce RNA is contamination by ribonuclease, here presents two protiocols for the
removal of contaminating DNA
Reagents required
1. PBS
2. 400 µl Lysis buffer
3. 4 µ of 20% SDS
4. 2.5 µl of 20 mg/ml proteinase K
5. 400 µl of 25:24:1 phenol/chloroform/isoamyl alcohol
6. 400 μl of 24:1 chloroform/isoamyl alcohol.
7. 1ml of 95% ethanol
8. 40 µl of 3 M sodium acetate (DEPC-treated)
9. 1 ml 80% ethanol /25% 0.1 M sodium acetate
10. 50 μl TE buffer
11. Mixture X containing DNAse
12. 25 μl DNase stop mixture
13. 325 μl of 100% ethanol
14. LiCl
Equipments Required
1. Centrifuge tubes
2. Table top centrifuge
3. Microcentrifuge
Approximate Time Required
Procedure
Prepare the cell sample from which RNA to be isolated
For monolayer cells:
1. Rinse cell monolayer twice with ice-cold PBS, and the cells are scraped to a
beaker containing ice cold PBS and transfer the mixture to a centrifuge tube
2. Centrifuge the solution at maximum speed for 15 sec in a microcentrifuge
maintaining temperature at 4°C and collect the cells.
For Cell Grown in Suspension
1. Harvests the cell by centrifugation in a table top centrifuge for 5-10 minutes at
room temperature
2. Remove the medium and resuspend the cells in one-half original culture volume
ice-cold PBS. Repeat the process
The Process
1. Transfer the cell to micro centrifuge tube containing 400 µl ice cold Lysis buffer
and place it on ice for 5 minutes.
2. Centrifuge the mixture in a microcentrifuge for 2 minutes at maximum speed.
3. Collect the supernatant in a clean tube and add 4 µl of 20% SDS to denature the
protein part. Mix immediately by vortexing.
4. To digest the protein part, add 2.5 µl of 20 mg/ml proteinase K. Incubate 15 min
at 37°C.
5. Add 400 µl of 25:24:1 phenol/chloroform/isoamyl alcohol, room temperature.,
mix thoroughly and collect in a centrifuge tube
6. The resultant mixture is centrifuged for 3 minutes in a microcentrifuge at
maximum speed .Carefully remove the supernatant to a clean tube and repeat the
step 7
7. Collect the aqueous upper phase to a clean tube and add 400 μl of 24:1
chloroform/isoamyl alcohol. Mix thoruogly by vortexing 15 to 30 sec
8. Centrifuge the mixture for 1 minute in a microcentrifuge at maximum speed, in
room temperature and collect the upper phase into a clean tube
9. Add 1 ml of 95% ethanol and 40 µl of 3 M sodium acetate (DEPC-treated) to the
RNA sample contained in a 1.5 ml microcentrifuge tube, invert to mix, and
incubate in an ice-water bath for at least 10 minutes. Store the sample at -20°C
overnight .
10. Centrifuge at maximum speed in a microcentrifuge for 15 min at 4°C, decant the
supernatant, and drain inverted on a paper towel
11. Add 1 ml 80% ethanol /25% 0.1 M sodium acetate to the pellet and rinse the
mixture.
12. Store at -20°C until needed.
Methods to Remove DNA Contamination from RNA Samples (2 Methods)
1. DNase digestion
2. Lithium chloride (LiCl) precipitation
DNase digestion
1. Redissolve the RNA in 50 μl TE buffer, pH 7.4.
2. Prepare the Mixture X containing DNAse
3. Add 50 μl of this Mixture to each RNA sample. Mix and incubate 15 min at 37°C.
4. Add 25 μl DNase stop mixture to stop the DNase reaction
5. Extract once with phenol/chloroform/isoamyl alcohol and once with
chloroform/isoamyl alcohol.
6. Add 325 μl of 100% ethanol and precipitate 15 to 30 min on ice or overnight at
−20°C.
Lithium chloride (LiCl) precipitation
1. Add LiCl to the Contaminated RNA solution to make up a final concentration of
at least 2 M.
2. Incubate on ice overnight and then collect the precipitated RNA by centrifugation
at full speed in a microcentrifuge for 15 min.
3. Remove the supernatant and wash the pellet with 1 ml of 3 M Sodium Acetate to
remove any small amounts of contaminating DNA.
4. Centrifuge the solution at full speed in a microcentrifuge for 15 min. Discard the
supernatant and remove the Sodium acetate by washing the pellet with -20°C
chilled 70% Ethanol.
5. Centrifuge the solution at full speed in a microcentrifuge for 15 min. Dry the
RNA pellet under vacuum for 90 sec and resuspend the RNA in sterile ddH2O.
Figures
Expected Results
Background information
1. RNases are very stable enzymes and generally require no cofactors to function.
Therefore, a small amount of RNase in an RNA preparation will create a real
problem.
Recipe
Mixture X containing DNAse
1. 10 μl of 100 mM MgCl2/10 mM DTT
2. 0.2 μl of 2.5 mg/ml RNase-free DNase
3. 0.1 μl of 25 to 50 U/μl placental ribonuclease inhibitor or vanadyl-ribonucleoside
complex,
4. 39.7 μl TE buffer
DNase stop mixture
1. 50 mM EDTA
2. 1.5 M sodium acetate
3. 1% (w/v) sodium dodecyl sulfate (SDS)
Lysis buffer
1. 50 mM Tris⋅Cl, pH 8.0 (APPENDIX 2)
2. 100 mM NaCl
3. 5 mM MgCl2
4. 0.5% (v/v) Nonidet P-40
TE Buffer
1. 10 mM Tris-Cl, pH 7.4, 7.5, or 8.0
2. 1 mM EDTA, pH 8.0
Trouble shooting
1. Any water or salt solutions used in RNA preparation should be treated with the
chemical diethylpyrocarbonate (DEPC). This chemical inactivates ribonucleases
by covalent modification
Products Available
1. PBS
2. Lysis buffer
3. SDS
4. Proteinase K
5. 24:1 chloroform/isoamyl alcohol
6. sodium acetate
7. TE buffer
Companies Supplying Products
Reference
Barlow JJ, Mathias AP, Williamson R, Gammack DB. A Simple Method for the
Quantitative Isolation of Undegraded High Molecular Weight Ribonucleic Acid.
Biochem. Biophys. Res. Commun. 1963: 13:61-66.
Cathala G, Savouret J, Mendez B, West BL, Karin M., Martial, JA., Baxter JD. A
Method for Isolation of Intact, Translationally Active Ribonucleic Acid. DNA 1983
:2:329-335.