OHS017
Risk assessment completed by:P Peake
OHS Risk Assessment and
Control Form
School/Unit: nephrology
Faculty/Division: Medicine
Document number
Initial Issue date
15.5.2010
Current version
1.0
Current Version
Issue date 15.5.2010
Next review date
15.5.2010
RRNAEXT.RA
For additional information refer to the OHS Risk Assessment and Control Procedure, the OHS Risk Rating Procedure and the Hierarchy
of Risk Controls.
Risk Assessment title: Extraction and purification of RNA from cells or tissues using TRI REAGENT (Sigma).
Step 1: Identify the activity
Describe the activity:
1A. Tissue: Homogenize tissue samples in TRI Reagent (1ml per 50–100 mg of tissue) in a Polytron or other
appropriate homogenizer.
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.
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–
2. Phase Separation:
To ensure complete dissociation of nucleoprotein complexes, allow samples to stand for 5 minutes at room
temperature. Add 0.1 ml of1-bromo-3-chloropropane or 0.2 ml of chloroform per ml of TRI Reagent used. 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).
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
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Date Effective: 15.5.2010
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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.
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 RNAase-free water to the RNA pellet. To facilitate dissolution, mix by repeated pipetting with a
micropipette at 55–60 °C for 10–15 minutes.
Describe the location:
Fume Hood
And Laboratory Rm 501 in Wallace Wurth
Step 2: Identify who may be at risk by the
activity
A number of people may be at risk from any activity. This may affect the risk controls needed. These people may include fellow workers, visitors, contractors
and the public. The location of the activity may affect the number of people at risk.
Operator and people nearby may be at risk from splash, spill or fumes from chemical reagents.
Steps 3 to 7: Identify the hazards, risks, and
rate the risks
1. An activity may be divided into tasks. For each task identify the hazards and associated risks.
2. List existing risk controls and determine a risk rating using the UNSW Risk Rating Procedure.
3. Additional risk controls may be required to achieve an acceptable level of risk. Re-rate the risk if additional risk controls used.
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Tasks
Hazards
Associated risks
(Step 3)
(Step 4)
Risk rating with
existing controls
*
Additional risk
controls
required
(Step 5)
(Step 6)
Existing risk controls
C
Homogenize
tissue samples
in TRI Reagent
using a
Polytron.
Exposure to
TRI Reagent
Polytron
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.
Liquid handling and
homogenization in fume
hood.
Standard PPE lab coat,
goggles and double gloves.
4
L
E
R
(Apply the
hierarchy of
risk controls)
Risk Rating with
additional controls *
(Step 7)
C
L
R
M
Homogenization in tall 15ml
tubes to reduce danger of
splash.
Training
Homogenization in tall 15ml
tubes to avoid blade
contact.
Electrical equipment
testing.
Polytron or other
homogenizer
Electrical appliance –
electrocution hazard
Spinning blade physical injury hazard
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Lyse cells
directly on the
culture dish.
After addition
of the reagent,
the cell lysate
should be
passed several
times through a
pipette to form
a homogenous
lysate.
Isolate cells
by
centrifugation
and then lyse
in TRI Reagent
by repeated
pipetting.
Exposure to
TRI Reagent
Exposure to
TRI Reagent
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.
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.
Liquid handling and
homogenization in fume
hood.
4
E
M
4
E
M
Standard PPE lab coat,
goggles and double gloves.
Training
Liquid handling and
homogenization in fume
hood.
Standard PPE lab coat,
goggles and double gloves.
Training
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2. Phase
Separation:
Add
chloroform,
cover the
sample
tightly, shake
vigorously.
Centrifuge the
resulting
mixture at
12,000g for 15
minutes at 2–8
°C.
Exposure to
TRI Reagent
Exposure to
chloroform
Centrifugation
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.
Chloroform
Toxic by inhalation,
in contact with skin
and if swallowed.
Carcinogenic and
mutagenic
Liquid handling in fume
hood.
4
E
M
Standard PPE lab coat,
goggles and double gloves.
Homogenization in tall 15ml
tubes to reduce danger of
splash.
Electrical equipment
testing.
Training
Note: Chloroform rapidly
degrades Latex and Nitrile
gloves . In the event of a
spill replace both layers
of gloves immediately.
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Transfer the
aqueous phase
to a fresh
tube and add
isopropanol.
Centrifuge at
12,000g for10
minutes at 2–8
°C.
Exposure to
TRI Reagent
Exposure to
chloroform
Exposure to
isopropanol
Centrifugation
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.
Chloroform
Toxic by inhalation,
in contact with skin
and if
swallowed.Carcinogenic
and mutagenic
Wash the RNA
pellet by
adding 75%
ethanol.
Vortex the
sample and
then
centrifuge at
7,500 g for 5
minutes at 2–8
°C. Remove
supernatant.
Exposure to
ethanol
Isopropanol (Propan-2ol 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.
Ethanol
Flammable liquid and
vapor. May cause central
nervous system depression.
Causes severe eye irritation.
Causes respiratory tract
irritation. Causes moderate
skin irritation.
Liquid handling in fume
hood.
4
E
M
2
D
L
Standard PPE lab coat,
goggles and double gloves.
Homogenization in tall 15ml
tubes to reduce danger of
splash.
Electrical equipment
testing.
Training
Note: Chloroform rapidly
degrades Latex and Nitrile
gloves . In the event of a
spill replace both layers
of gloves immediately.
Avoid flames. Only small
volumes of Isopropanol
stored on bench.
Liquid handling in fume
hood.
Standard PPE lab coat,
goggles and double gloves.
Training
Avoid flames. Only small
volumes of Isopropanol
stored on bench.
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Current Version: 15.5.2010
Briefly dry the
RNA pellet for
5–10 minutes by
air drying. Add
an appropriate
volume of
RNAase-free
water to the
RNA pellet. To
facilitate
dissolution,
mix by repeated
pipetting with
a micropipette
at 55–60 °C for
10–15 minutes.
Use of a
55–60 °C
Heating
block
Electrical equipment
shock
Burn
* C = consequence
Electrical equipment
testing.
2
D
L
Training
L = likelihood R = risk rating
from the UNSW Risk Rating Procedure
Step 8 Documentation and supervisor
approval
Completed by: (name)
P PEAKE (signature)
Authorised by:
(signature)
Date:
Step 9: Implement the additional risk
controls identified
Indicate briefly what additional risk controls from Step 6 above were implemented, when and by whom.
Risk control:
Date:
Implemented by:
Risk control:
Date:
Implemented by:
Step 10: Monitor and review the risk
controls
It is important to monitor risk controls and review risk assessments regularly. Review is required when there is a change in the process, relevant
legal changes, and where a cause for concern has arisen. Reviews could be scheduled on an annual basis. If the risk assessment has substantially
changed a new risk assessment is warranted.
Review date:
Reviewed by:
Authorised by:
Documentation
It is a requirement that legal and advisory documentation that supports this risk assessment be listed. Such documentation includes Acts,
Regulations, Australian Standards and Codes of Practice, where applicable.
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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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Risk Assessment and Control Form
Date Effective: 15.5.2010
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Current Version: 15.5.2010
UNSW Concise OHS Risk Rating Table
OHS697
What you need to do
1. Consider what can go wrong that can hurt someone
2. Determine what the most likely outcome would be - Consequences
3. Determine how likely those consequences are - Likelihood
4. Calculate the risk rating
5. Required action
CONSEQUENCES:
How severely could someone be hurt
death or permanent disability to one or more persons
hospital admission required
medical treatment required
first aid required
injuries not requiring first aid
Severe
Major
Moderate
Minor
Insignificant
How likely are those consequences?
expected to occur in most circumstances
will probably occur in most circumstances
could occur at some time
is not likely to occur in normal circumstances
may occur only in exceptional circumstances
LIKELIHOOD:
Almost certain
Likely
Possible
Unlikely
Rare
CONSEQUENCES
Insignifica
nt
1
Minor
2
Moderate
3
Major
4
Severe
5
M
H
H
VH
VH
M
M
H
H
VH
Possible
C
L
M
H
H
VH
Unlikely
D
L
L
M
M
H
Rare
E
L
L
M
M
M
LIKELIHOOD
Almost
certain
A
Likely
B
Risk level
Very high
High
Medium
Low
Required action
Act immediately:
The proposed task or process activity must not proceed. Steps must be taken to lower the risk level to as
low as reasonably practicable using the hierarchy of risk controls.
Act today:
The proposed activity can only proceed, provided that:
(i) the risk level has been reduced to as low as reasonably practicable using the hierarchy of
risk controls;
(ii) the risk controls must include those identified in legislation, Australian Standards, Codes of
Practice etc.
(iii) the risk assessment has been reviewed and approved by the Supervisor and
(iv) a Safe Working Procedure or Safe Work Method has been prepared.
(v) The supervisor must review and document the effectiveness of the implemented risk
controls.
Act this week:
The proposed task or process can proceed, provided that:
(i) the risk level has been reduced to as low as reasonably practicable using the hierarchy of
risk controls;
(ii) the risk assessment has been reviewed and approved by the Supervisor and
(iii) a Safe Working Procedure or Safe Work Method has been prepared.
Act this month:
Managed by local documented routine procedures which must include application of the hierarchy of
controls.
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UNSW Concise OHS Risk Rating Table
Effective date: 01/01/2007
Uncontrolled document when printed
Current Version: 2.6,16/07/2008