OHS026
Safe Work Procedure
Faculty/Division
Medicine
Document number
SOMS GBD SWP 021
School/ Divisional Unit
School of Medical Sciences
Initial Issue date
13-03-2009
Current version
1.0
Current Version
Issue date
20-04-2010
Next review date
20-04-2013
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
RNA extraction
Title:
Description: extract RNA from human or animal tissue
Associated risk assessment title and location: RA Handling of human tissue 020, RA bench top centrifuge 003, RA waste
management 015 located in Room 209B
Describe the activity or process
Epicentre method:

Prepare eppendorf tubes with hole pierced in lid using a new 27 gauge needle.

Weigh sample and place them in the tubes containing cell lysis solution with proteinase K, chop up with scissors and
homogenize with biopsy homogenizer.

Incubate at 65oC for 15min, add MPC protein precipitation reagent and then centrifuge at 12,000g for 15min.

Precipitate nucleic acid with isopropanol, leave at –20oC for 2hrs, centrifuge for 10min at 4oC and wash pellets with 75% of
ethanol twice.

Digest DNA with DNase in the presence of RNasin to protect RNA at 37OC for 30min and then repeat step of MPC and
isopropanol.

RNA pellets are dissolved in RNase free water and amounts pf RNA extracted are measured by spectrophotometer.
TRIzol method:

Weigh samples (100mg) and place them in 1ml Trizol reagent in a special centrifuge tube for GeneWorks homogenizer.

Homogenize the samples and leave for 5min.

Add 0.1ml of chloroform, cap sample tubes securely and shake thoroughly.

Centrifuge the sample at 12,000g for 15min at 4oC.

Transfer the aqueous phase to a fresh tube, precipitate RNA with isopropanol at –20oC for 2hrs, centrifuge 10,000g for
15min at 4oC and wash the pellets with 75% ethanol.

Remove trace DNA contamination by incubating RNA sample with DNase in the presence of RNAsin at 37 oC for 30min.

Add 2xT&C Lysis buffer and MPC protein precipitation reagent and centrifuge at 12,000g for 15min.

Precipitate DNA free RNA with isopropanol, leave at –20oC for 2 hrs, centrifuge at 12,000g for 15min and wash the pellets
with 75% ethanol.

RNA pellets are dissolved in RNase free water and amounts of RNA extracted are measured by spectrophotometer.
Electrophoresis to check the integrity of RNA (some procedures are performed in fume hood):

Heat 1g agarose in 72ml of water until dissolved, and then cool to 60oC.

Add 10ml of 5x MOPS running buffer and 18ml of formaldehyde.

Pour the gel onto a tray with a comb that form wells large enough to accommodate at least 25l.

Remove the comb, assemble the gel in the tank, add enough 1x MOPS running buffer to cover the gel by a few of
millimeters.

To 1mg of RNA, add 0.5 volumes formaldehyde, heat denature samples at 65oC for 5-15min.

Add loading dye and ethidium bromide.

Load the gel and electrophorese at 80V until the bromophenol blue has migrate at least 2-3cm into the gel.

Visualize the gel on the Gel Doc with UV translluminator/filter 1.
List all resources required including plant,
chemicals, personal protective clothing and
equipment, etc






Suitable PPE
RNA extraction Kit, DNase, RNasin
Chloroform, isopropanol, 75% ethanol
Formaldehyde, ethidium bromide, agarose , MOPS running buffer
Centrifuge tubes, samples, scissors, needles, 70% ethanol spray bottle
Spectrophotometer, homogenizer and centrifuge
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Page 1 of 2
Safe Work Procedure
Date Effective: 01/01/2007
Uncontrolled document when printed
Current Version: 1.2, 15/08/2007
List potential hazards and risk controls including
specific precautions required





Chemicals may be hazardous – for chemicals, check MSDS and wear PPE, use fume hood if necessary, all users are well
trained and all spills are cleaned immediately and disposed in chemical waste. All liquid chemical waste is collected into a
special waste bottle.
Tissue can be contaminated or infectious – training is required if handling human tissue, wear PPE, all spills are cleaned
immediately with 70% ethanol and disposed in biological waste. Scissors and spatula is wiped with 70% ethanol and then
washed with water. All weight boats and tubes are disposed in biological or autoclaved waste.
Potential electrical hazard – do annual inspections of electrical equipment and ensure electrical safety tag is current.
Potential aerosol hazard – wear PPE and use a biosafety hood for using formaldehyde and ethidium bromide.
UV light may be hazardous – use a UV protective face shield if it is necessary and cover the gel when it is transported.
List emergency shutdown instructions

Centrifuge and spectrophotometer have “OFF” switch that can be used in emergency.

Shut all electrical equipment off at power point in case of emergency.
List clean up and waste disposal requirements
For chemicals:
Clean up with detergent and then with water
Dispose into chemical waste
For human sample:
Clean up or sock with 70% ethanol
Dispose into autoclaved waste
List legislation, standards and codes of practice used
in the development of the SWP
NSW OHS Act 2000
NSW OHS Regulation 2001
Australia Dangerous Goods Code
Code of Practice for the Labeling of Workplace Substances
AS/NZS 2243.2:2006. Safety in laboratories. Part 2: Chemical aspects
Australian Standard AS2243.6-1990. Safety in laboratories. Part 6: Mechanical Aspects.
Australian Standard AS2243.7-1991. Safety in laboratories. Part 7: Electrical Aspects.
AS/NZS 2161.1:2000 Occupational Protective Gloves – Selection, Use and Maintenance
Safe Work Procedure Form (OHS026)
UNSW Hazardous Waste Disposal Procedure
Supervisory approval, training, and review
Supervisor: Prof E Burcher
Signature:
Plant custodian:
Signature
List competency required – qualifications, certificates, licensing, training - e.g. course or instruction:
SWP review date: 20-04-2013
Responsibility for SWP review: Fei Shang
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Page 2 of 2
Safe Work Procedure
Date Effective: 01/01/2007
Uncontrolled document when printed
Current Version: 1.2, 15/08/2007