THE UNIVERSITY OF NEWCASTLE- DISCIPLINE OF MEDICAL BIOCHEMISTRY. STANDARD OPERATING PROCEDURE PROCEDURE NO: GLP 009 MOD: 1st Issue Page: 1 of 9

Transcription

1 Page: 1 of 9 1. Risk Assessment: This risk assessment has been prepared using the National Safety Council of Australia Risk Score Calculator and risks have been determined to be as stated. This Risk Assessment is to be used as a general guide and as such, cannot accommodate all the varying factors that may be encountered when using this equipment. Therefore, personnel are requested to conduct their own Risk Assessment before using this equipment to include any extra hazards introduced by the task performed. HAZARDS 1. Sodium Hydroxide (harmful, causes burns) 2. Ammonium Persulphate (harmful, causes burns) 3. Glycerol (irritant) 4. Formamide (harmful, irritant) 5. Ethanol (flammable) 6. Boric Acid (harmful, irritant) RISK ASSESSMENT 1. Risks are associated with using the CyScribe Post-labelling Kit (irritant) RISK CONTROL 1. All chemicals should be considered as potentially hazardous. Wear suitable protective clothing, ie. lab coat, safety glasses and gloves. 2. All steps should be performed in the fume hood. 2. Purpose: 2.1 Gene Expression 3. Equipment: 3.1 Heating Blocks WRITTEN BY CHECKED BY AUTHORISED BY NAME (signed) Rowan Foster DATE 2 nd April 2003 Distributed To: GLP Master File / GLP Lab File

2 Page: 2 of Bench top centrifuge o C Freezer o C Incubator 3.5 Array Scanner 3.6 Computer micron filters 3.8 UV visible spetrophotometer 3.9 Various pipettes 4. Materials: 4.1 Sterile amber eppendorf tubes 4.2 Sterile plugged tips 4.3 Foil 4.4 Gloves 4.5 Nuclease free water (Gibco) 4.6 Cy-Scribe Post labelling Kit (Amersham) M NaOH M HEPES free acid Quiagen PCR DNA Purification Kit M Sodium Bicarbonate M Hydroxylamine Hydrochloride Absolute ethanol M sodium acetate % ethanol oligonucleotide da RNA x SSC % SDS 4.7 Hybridisation chamber 4.8 slides

3 Page: 3 of 9 5. Set Up: 5.1 Photocopy the Batch Record attached to this SOP (Illustration 10.1). Ensure the Batch Record is completed as the procedure is carried out (if required). 5.2 Ensure that you have read and understood the Safety Precautions (Section 6 of this SOP) prior to commencing this procedure. Sign the Batch Record to indicate that this has been done. 5.3 This procedure can be done in two days. However its' best done over three; stopping after EtOH precipitation (7.3.16), hybridising overnight, washing & scanning on day Safety Precautions: 6.1 Good laboratory techniques are to be used at all times. 7. Method: NOTE: Do all steps in a Fume Hood 7.1 Labelling first-strand cdna with amino allyl-dutp Set heating blocks to 70C and 42C Get out the Anchored oligo(dt), water and trna and place on ice to thaw Add 25ug trna, 3uL Anchored oligo(dt) and water to a total volume of 11uL Mix gently by pipetting up and down Incubate reactions at 70C for 5 minutes Cool reactions at room temperature for 10 minutes Get out the 5x CyScript buffer, 0.1M DTT, Nucleotide mix, AA-dUTP and place on ice Spin down reactions for 15 seconds in a microcentrifuge

4 Page: 4 of Extension reaction Add; 4uL of 5x CyScript buffer, 2uL of 0.1M DTT, 1uL of Nucleotide mix, 1uL of AA-dUTP and 1uL of CyScript reverse transcriptase (enzyme) Mix by stirring with a pipette tip Incubate reactions at 42C for 1.5 hours 7.3 Purification of amino allyl modified cdna Adjust water bath to 37C Add 2uL 2.5M NaOH to each cdna reaction Mix reactions by vortexing and spin for 15 seconds in a microcentrifuge Add 10uL 2M HEPES free acid to each reaction Mix reactions by vortexing and spin for 15 seconds in a microcentrifuge Add 3uL 3M Sodium Acetate to each cdna sample Add 75uL of absolute ethanol Mix reactions by vortexing and spin for 15 seconds in a microcentrifuge Incubate at -80C for 1 hour Microcentrifuge at 13,000rpm for 30 minutes at room temperature Remove supernatant without disturbing the pellet Add 1mL of 70% EtOH to each sample Microcentrifuge at 13,000rpm for 15 minutes at room temperature Remove supernatant without disturbing the pellet Air dry the pellet and resuspend in 15uL of nuclease free water This can now be stored at -20C

5 Page: 5 of Labelling of amino allyl modified cdna with CyDye Resuspend one aliquot of CyDye (Cy3 or Cy5 as supplied in kit) in 15uL of fresh 0.1M Sodium Bicarbonate ph9.0 (stored at -20C, throw after use) Immediately add one aliquot of resuspended CyDye to one tube of resuspended amino allyl modified cdna Mix by stirring and incubate at room temperature, in the dark, for 1 hour Add 15uL of 4M Hydroxylamine to each coupling reaction Mix by stirring and incubate at room temperature, in the dark, for 15 minutes 7.5 Purification of CyDye-labelled cdna As per Qiagen kit Elute CyDye-labelled cdna from the column with 50uL of nuclease free water Take 2uL of CyDye-labelled cdna and add to 98uL of nuclease free water Blank spectrophotometer, at each wavelength, with same nuclease free water Measure the absorbance of Cy3 at 550nm and Cy5 at 650nm The amounts of Cy3 and Cy5 incorporated into cdna can be calculated: pmoles Cy3 or Cy5 in measured sample = A x Z x dilution factor x E where: A = absorbance Cy3 at 500nm or Cy5 at 650nm E = extinction coefficient for Cy3 (150,000) or Cy5 (250,000) Z = volume of cdna (48 x 10-6 ) Incorporation of dye = pmol of CyDye in sample ug of cdna in the measured volume (48uL)

6 Page: 6 of Hybridisation of Slides Note: Pre-warm incubator to 42C and heating block to 95C Combine 15pmol of each CyDye into one tube Dry down the cdna solution in a rotary evaporator, in an amber microcentrifuge tube. Protect the tubes from light Dissolve the cdna in 6uL of nuclease free water Denature the cdna by heating at 95C for 2 minutes Cool the cdna solution on ice for 30 seconds Add 1.5uL oligonucleotide da80 and mix well Incubate the mixture at 75C for 45 minutes Add 7.5uL of hybridisation buffer (look in kit) and 15uL of 100% Formamide Mix well and spin for 15 seconds in a microcentrifuge Pipette hybridisation mixture onto microarray slide Hybridise at 42C for hours in a humid hybridisation chamber 7.7 Washing Hybridised Slides Pre-warm 1x SSC, 0.2% SDS and 0.1x SSC, 0.2% SDS to 55C Wash hybridised slides with 1x SSC, 0.2% SDS pre-warmed to 55C, for 10 minutes, at room temperature using a platform shaker. Remove cover slip only after the slide has been immersed in the washing solution Follow with 2x 10 minute, room temperature, washes in 0.1x SSC, 0.2% SDS pre-warmed to 55C Wash slides 2x 1 minute room temperature 0.1x SSC Dip slides into distilled water for 5 seconds maximum Immediately dry the slides with gentle air stream Scan the slides

7 Page: 7 of 9 8. Maintenance: 8.1 Sodium bicarbonate ph9.0 must be thrown out after 3 months 9. Shutdown: 9.1 Attach scan picture to batch record and note file name 10. Illustrations: See attached 10.1 Batch Record 11. Check List: 11.1 Ensure batch record documentation is complete. 12. References: 12.1 Amsersham CyScribe Post-labelling Kit Instruction manual 13. Change History: 13.1 Issue Number: 1st Issue Date Issued: 13.2 Issue Number: Date Issued: Reason for Change:

8 Page: 8 of 9 BATCH RECORD Page: 1 of 2 ILLUSTRATION 10.1 BATCH RECORD FOR MICROARRAY Date Kit batch Cy3 Cy5 Problems? record # reading reading Comments: Batch Record Completed By: Date: / / Countersigned:

9 AUSTRALIAN INSTITUTE OF MUCOSAL IMMUNOLOGY PTY LTD Page: 9 of 9 BATCH RECORD Page: 2 of 2 BATCH RECORD FOR MICROARRAY Date Kit batch Cy3 Cy5 Problems? record # reading reading Comments: Batch Record Completed By: Date: / / Countersigned: