OHS026
Safe Work Procedure
Faculty/Division
Medicine
Document number
RRNAEXT.SW
School/ Divisional Unit
nephrology
Initial Issue date
Current version Current Version
Issue date
15.5.2010
Next review date
15.5.2013
The Writing Safe Work Procedures Guideline (OHS027) should be consulted to assist
in the completion of this form.
Safe Work Procedure Title and basic
description
Title:
RNA extraction using TRIreagent
Description: Extraction of RNA from tissues or cells using the pre-prepared phenol/guanadinium
thiocyanate mix TRIreagent (SIGMA)
Associated risk assessment title and location:
Describe
the activity or process
These procedures involve the use of toxic and flammable materials. PPE must include lab
coat, goggles and double gloves. The handling of RNALater and TRIReagent must be in a
fume hood. During centrifuge spins take care not to contaminate centrifuge rotors and
other surfaces with spilled TRIReagent. In the event of a spill use a spill kit. For minor
splashes, remove immediately with tissues and dispose of in a sealed
NOTE 1: TRI Reagent is stable for at least 12 months stored at 4˚C.
NOTE 2: It is important to protect RNA from degradation by RNases. This can be done by using
sterile plasticware, DEPC–treated and autoclaved solutions, pipettes reserved for RNA work, and
by wearing disposable gloves. Glassware should be oven baked at 200oC
NOTE 3: If using tissues, immediately place in RNAlater (Ambion) after collection (see list item 1
below for safety guidelines). RNA is stable in this solution for several hours at room temperature.
If keeping overnight, store at 4oC. If keeping longer, store at 4oC overnight and then at -20oC or 80oC after. Try not to store for too long as the dehydrating nature of the RNALater reduces the
efficiency of RNA extraction and yield will reduce.
1A. Tissue:Homogenize tissue samples in TRI Reagent (1ml per 50–100 mg
of tissue) in a Polytron or other appropriate homogenizer. See item 2
below for safety guidelines.
Note: If minimal shearing of the DNA is desired, use a loosely fitting
homogenizer, not a Polytron (see DNA Isolation, step 3, note b). The
volume of the tissue should not exceed 10% of the volume of the TRI
Reagent.
1B. Monolayer cells:Lyse cells directly on the culture dish. Use 1 ml of
the TRI Reagent per 10 cm2 of glass culture plate surface area. After
addition of the reagent, the cell lysate should be passed several times
through a pipette to form a homogenous lysate.
Note: TRI Reagent is not compatible with plastic culture plates.
(However, this can be done if you use larger volumes of TRI Reagent and
work rapidly SP e.g. 3-5mls reagent per 10cm diameter dish).
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1C. Suspension cells: Isolate cells by centrifugation and then lyse in
TRI Reagent by repeated pipetting. One ml of the reagent is sufficient
to lyse 5–10x106 animal, plant, or yeast cells, or 107 bacterial cells.
Notes:
a. If samples have a high content of fat, protein, polysaccharides, or
extracellular material such as muscle, fat tissue, and tuberous parts of
plants an additional step may be needed. After homogenization,
centrifuge the homogenate at12,000g for 10 minutes at 2–8°C to remove
the insoluble material (extracellular membranes, polysaccharides, and
high molecular mass DNA). The supernatant contains RNA and protein. If
the sample had a high fat content, there will be a layer of fatty
material on the surface of the aqueous phase that should be removed.
Transfer the clear supernatant to a fresh tube and proceed with step 2.
Recover the high molecular mass DNA from the pellet by following DNA
Isolation, steps 2 and 3.use a loosely fitting homogenizer, not a
Polytron (see DNA Isolation, step 3, note b). The volume of the tissue
should not exceed 10% of the volume of the TRI Reagent. Recover the high
molecular mass DNA from the pellet by following DNA Isolation, steps 2
and 3.
b. Some yeast and bacterial cells may require a homogenizer.
c. After the cells have been homogenized or lysed in TRI Reagent,
samples can be stored at –70 °C for up to 1 month.
2. Phase Separation:
To ensure complete dissociation of nucleoprotein complexes, allow
samples to stand for 5 minutes at room temperature. Add 0.1 ml of1bromo-3-chloropropane or 0.2 ml of chloroform (see Phase Separation,
notes a and b) per ml of TRI Reagent used (see item 3 below for
chloroform safety guidelines). Cover the sample tightly, shake
vigorously for 15 seconds, and allow to stand for 2–15 minutes at room
temperature. Centrifuge the resulting mixture at 12,000g for 15 minutes
at 2–8 °C. Centrifugation separates the mixture into 3 phases: a red
organic phase (containing protein), an interphase (containing DNA), and
a colorless upper aqueous phase (containing RNA).
Notes:
a. 1-Bromo-3-chloropropane is less toxic than chloroform and its use for
phase separation decreases the possibility of contaminating RNA with
DNA.
b. The chloroform used for phase separation should not contain isoamyl
alcohol or other additives.
c. For isolation of poly A+ fraction from the aqueous phase see Appendix
I.
RNA Isolation
1. Transfer the aqueous phase to a fresh tube and add 0.5 ml of isopropanol (see item 4 for
safety guidelines) per ml of TRI Reagent used in Sample Preparation, step 1 and mix.
Allow the sample to stand for 5–10 minutes at room temperature. Centrifuge at 12,000g
for10 minutes at 2–8 °C. The RNA precipitate will form a pellet on the side and bottom of
the tube.
2. Remove the supernatant and wash the RNA pellet by adding a minimum of 1 ml of 75%
ethanol (see item 5 for safety guidelines) per1 ml of TRI Reagent used in Sample
Preparation, step 1. Vortex the sample and then centrifuge at 7,500 g for 5 minutes at 2–8
°C.
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Notes:
a. If the RNA pellets float, perform the wash in75% ethanol at
12,000g.
b. Samples can be stored in ethanol at 2–8 °C for at least 1 week and
up to 1 year at –20 °C.
3. Briefly dry the RNA pellet for 5–10 minutes by air drying or under
a vacuum. Do not let the RNA pellet dry completely, as this will
greatly decrease its solubility. Add an appropriate volume of RNAasefree water to the RNA pellet. To facilitate dissolution, mix by
repeated pipetting with a micropipette at 55–60 °C for 10–15 minutes.
Notes:
a. Final preparation of RNA is free of DNA and proteins. It should
have a 260/280 ratio of 1.7.
b. Typical yields from tissues (mg RNA/mg tissue):liver, spleen, 6-10
mg; kidney, 3-4 mg; skeletal muscle, brain, 1-1.5 mg; placenta, 1-4
mg.
c. Typical yields from cultured cells(mg RNA/106 cells): epithelial
cells, 8-15 mg; fibroblasts, 5-7 mg.
d. Ethidium bromide staining of RNA in agarose gels visualizes two
predominant bands of small (2 kb) and large (5 kb) ribosomal RNA, low
molecular mass (0.1–0.3 kb) RNA, and discrete bands of high molecular
mass (7–15 kb) RNA.
List all resources required including
plant, chemicals, personal protective
clothing and equipment, etc
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RNALater (Ambion)
TriReagent (Sigma Aldrich)
Chloroform
Isopropanol
Ethanol
EDTA
Tris-HCl
RNase/DNase Free H2O
Refrigerated microcentrifuge
Spectrophotometer or NanoDrop
Fume cabinet
Polytron or other homogenizer
Refrigerated centrifuge capable of 12,000g for quantities <30ml e.g. Sorval.
Lab coat
Safety goggles
Double gloves
List potential hazards and risk controls
including specific precautions required
1.RNALater (Ambion)
Inhalation : Causes respiratory tract irritation.
Skin : May cause skin irritation.. May be harmful if absorbed through skin.
Eyes : Causes eye irritation.
Ingestion : May be harmful if swallowed.
Specific Controls: Pipette in fume cupboard.
2.TRI Reagent
Toxic by inhalation, in contact with skin and if swallowed.
Harmful: contains phenol danger of serious damage to health by prolonged exposure through
inhalation in contact with skin, and if swallowed. Contact with acids liberates very toxic gas.
Causes burns. Possible risk of irreversible effects. Harmful to eyes. Harmful to aquatic organisms,
may cause long-term adverse effects in the aquatic environment.
Specific controls: handle in fume hood. Wear double gloves.
In the event of a spill on skin: flush with copious amounts of water for at least 15 minutes. Remove contaminated
clothing and shoes. Call a physician.
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3.Chloroform
Toxic by inhalation, in contact with skin and if swallowed.
Carcinogenic and mutagenic
Specific controls: handle in fume hood. Wear double gloves. Note: Chloroform rapidly degrades Latex
and Nitrile gloves . In the event of a spill replace both layers of gloves immediately.
4.Isopropanol (Propan-2-ol or 2-propanol).
Flammable liquid and vapor. May cause central nervous
system depression. Causes severe eye irritation. Causes respiratory tract irritation. Causes moderate skin irritation.
Specific controls: Large quantities (2.5L) stored in flame-proof cabinet. Only small quantities
(100mls) kept on bench
5.Ethanol
Flammable liquid and vapor. May cause central nervous
system depression. Causes severe eye irritation. Causes respiratory tract irritation. Causes moderate skin irritation.
Specific controls: Large quantities (2.5L) stored in flame-proof cabinet. Only small quantities
(100mls) kept on bench
6.Spectrophotometer
Electrical appliance – electrocution hazard
7.Refrigerated microcentrifuge
Electrical appliance – electrocution hazard
Spinning rotor- physical injury hazard
8.Polytron or other homogenizer
Electrical appliance – electrocution hazard
Spinning blade - physical injury hazard
9.Refrigerated centrifuge capable of 12,000g
Electrical appliance – electrocution hazard
Spinning rotor- physical injury hazard
List emergency shutdown instructions
For electrical appliances, the emergency cutoff switch (red button) is
located on the wall near the entrance to the lab.
TRI REAGENT SKIN CONTACT: Remove contaminated clothing and shoes
immediately. Wash affected area with soap or mild detergent and large
amounts of water until no evidence of chemical remains (e.g. TRI Reagent
odor). Use a deluge shower for at least 15 minutes. In case of chemical
burns, cover area with sterile, dry dressing, bandage securely, but not
too tightly. Get medical attention immediately.
List clean up and waste disposal
requirements
All solution s should be disposed of in chemical waste containers.
Solutions containing organic solvents i.e. phenol and chloroform should be put in a separate waste
container to the aqueous waste solutions .
Contaminated solid waste should be disposed of through the biological and chemical waste stream.
Small amounts of phenol can generate powerful fumes. Ensure that contaminated solid waste is
contained if placed into an open bin. A heat-sealed polypropylene bag works well.
List legislation, standards and codes of
practice used in the development of the
SWP
NSW OHS Act 2000
NSW OHS Regulation 2001
Code of Practice for the Labelling of Workplace Substances
AS/NZS 2243.2:2006. Safety in laboratories. Part 2: Chemical aspects
AS/NZS 2243.3-2002. Safety in laboratories. Part 3: Microbiological aspects and containment facilities.
AS/NZS 2243.4: 1998 Safety in Laboratories Part 4; Ionising Radiations
AS/NZS 2243.6-1990. Safety in laboratories. Part 6: Mechanical Aspects.
AS/NZS 2243.7-1991. Safety in laboratories. Part 7: Electrical Aspects.
AS/NZS 2161.1:2000 Occupational Protective Gloves – Selection, Use and Maintenance
AS/NZS 1336:1997 Recommended Practices for Occupational Eye Protection
UNSW Hazardous Waste Disposal Procedure
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Supervisory approval, training, and review
Supervisor: P PEAKE
Signature:
Plant custodian:
Signature
List competency required – qualifications, certificates, licencing, training - eg course or
instruction:
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Date Effective: 15.5.2010
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Current Version: 15.5.2010