RNA EXTRACTION FROM HCC TISSUE
Prepared by Aung Myat Oo and Shanthi Wasser
Please read the complete SOP, especially the safety considerations, before
starting on the experiments. Ensure that all reagents and equipment are
available in sufficient quantities before starting experiment.
General safety considerations
Human tissue collected from the Operating Theatre may contain HBV or HIV, which
is a BSL2 agent. However, HBV is not transmitted by the aerosol route and there is an
effective vaccine against it. All personnel involved in this project (including NUH
Operating Theatre and housekeeping personnel) will be prophylactically vaccinated
against HepB. Antibody status will be checked every 5 years and staff sent for revaccination/booster shots if required.
HepB waste: All consumables that come into contact with the human tissue will be
decontaminated in 10% bleach. As per Public Health Agency of Canada's MSDS
(http://www.phac-aspc.gc.ca/msds-ftss/msds76e.html), HBV can be decontaminated
with 1% sodium hypochlorite or 70% ethanol. Alternatively, decontamination can be
carried out with 1% VirKon®S solution. Refer to SOP on Biological Spill Cleanup for
detailed procedures.
Biohazard waste at Dept of Medicine is disposed of by NUH contract service
provider.
A. Instruments and reagents checklist
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Motorized homogenizer.
Disposable pellet pestles (autoclaved).
1.5 ml Eppendorf tubes (autoclaved) and stand.
Large Styrofoam cup filled with ice.
1000µl, 200µl and 100µl filtered pipette tips and pipettes.
Trizol
Chloroform
Isopropanol
75% ethanol
RNAse free water.
B. Protocol for RNA extraction
All procedures except centrifugation are to be carried out in the biological safety
cabinet.
1. Wear gloves, lab coat (long sleeve) and safety glasses. All procedures are to
be carried out in a Biological Safety Cabinet.
2. Place 500 µl of Trizol in Eppendorf tube and place on ice.
3. Place tissue in the tube of Trizol.
Trizol solution will inactivate any live HBV present in the tissue and render
them non-infective. However, Trizol reagent contains phenol which may cause
burns when in contact with the skin. Therefore this procedure must be carried
out in the BSC. Although BSCs are not to be used as replacements for fume
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cupboards, the use of the small amounts of Trizol required in this procedure
will not damage the HEPA filter of the BSC.
Please refer to Trizol MSDS (on file in the lab) for action to be taken in case
of accidental spills/splash of Trizol. ( NB: PEG required to “clean up”
accidental splashes on the skin is available in the lab chemical shelf).
4. Homogenize tissue with tube in ice using the motorized homogenizer. This
process must be carried out in the BSC cabinet to minimize dispersal of
aerosols that are formed during the process.
5. After homogenization, add another 500 µl of Trizol.
6. Incubate for 5 min at room temperature.
7. Add 200 µl of chloroform and shake tube by for 15 seconds
8. Incubate at room temperature for 3 minutes.
9. Centrifuge at 12,000 g for 15 min at 2-8 degree C.
10. Remove the upper layer and place in new tube.
11. Add 500 µl of isopropanol and incubate at room temp for 10 min.
12. Centrifuge at 12,000 g for 10 min at 2-8 degree C.
13. Remove the supernatant (leave the pellet alone).
14. Add 1 ml of ethanol.
15. Vortex (making sure that the tubes are securely capped to prevent accidental
spills) and centrifuge at 7500 g for 5 min at 2-8 degree C.
16. Remove supernatant and air dry the RNA pellets for 10 min.
17. Dissolved in 50-100 µl of RNAse free water and store at -80 freezer.
SAFETY MEASURE: Before preparation procedure, wear gloves, lab coat (long
sleeve) and eye protective gear (safety glasses). All processes of tissue cutting and
processing procedures are to be carried out in the biological safety cabinet. All
materials in contact with or contaminated with tissue were disposed of into designated
biohazard containers. All equipment and work surfaces were disinfected after use with
freshly diluted 10% bleach solution or 1% VirKon solution.
Accidental cut or stab with items contaminated with blood: Wash the wound with
soap and running water immediately and continue for at least 10 minutes. Squeeze or
scrub the wound to induce bleeding if possible. Report and get medical assistance.
For eye exposures, flush eyes under eyewash for at least 10 minutes. Report and get
medical assistance.
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