P-BAIR-3 - P-BAIR-3

Type
hybridization
Description
Hybridisation and Staining Protocols

These are the protocols used by the Microarray Centre to process labelled cRNA for GeneChip experiments, There are several protocols which can be used and their use is determined by the state of the cRNA and the type of chip being used.


1.Hybridisation of fragmented cRNA to GeneChip

2.Preparation of GeneChip Station

3.Either: a. Test 3 Processing b. Genome scale array processing

4.Shutdown of GeneChip Station


A. Hybridisation of fragmented cRNA to GeneChip


Things to note:

1. There are two septa on the back of the GeneChip. Whilst filling or emptying the hybridisation chamber through one septum, a pipette tip must be inserted into the other as a vent.

2. The more you inset/remove tips into the septa, the looser they will become. Eventually they will start to leak.


Method

1. Equilibrate array at room temperature for 30 minutes before use.

2. Heat frozen fragmented cRNA at 99oC for 5 minutes.

3. During incubation, pre-wet the array by filling with 200µl (100µl for test or mini arrays) of 1x hybridisation Buffer. Incubate in rotisserie oven at 45oC, 60rpm for 10 minutes.

4. Cool fragmented cRNA at 45oC for 5 minutes.

5. To bring any particulate matter to the bottom of the tube, spin fragmented cRNA at maximum speed (ý13,000g) for 5 minutes.

6. Remove buffer solution from array, ensuring that all solution is removed.

7. Add 200µl (100µl for test or mini array) of fragmented cRNA avoiding any debris at the bottom of the tube.

8. Hybridise in rotisserie oven at 45oC, 60rpm, for 16 hours (overnight).

9. Store remaining volume of fragmented cRNA at 20oC.

10. After hybridization, proceed to Preparation of GeneChip station.


B. Preparation of GeneChip station


Things to note:

1. Scanner must be switched on before launching Microarray Suite. When the scanner is switched on, yellow and green lights should illuminate. If there is a problem the red light will come on, or the yellow and green lights will flash. When the scanner is warmed the yellow light is extinguished.

2. When entering experiment info in step (4), complete experiment name, array type, array lot, operator name, and sample type (cRNA). If known fill in sample description and insert Target ID in sample project.

3. If a fluidics station has already been used and wash solutions have not been changed, steps (7-9) will not be necessary.

4. In step (7) take care not to touch the hoses.

5. In step (8) prime all ports of a fluidics station even if they are not all required.


Method

1. Switch on scanner.

2. Login to computer.

3. Launch Microarray Suite from desktop icon.

4. From Tools menu select Defaults and then File Locations to check the location in which the files will be saved.

5. Open Expts dialogue box. Complete as much information as possible and then save experiment. If performing wash and stain with antibody amplification, create 2 copies of each experiment. For one, mark the end of the experiment name with ba (before antibody); mark the other with aa (after antibody). Once all experiments have been entered close dialogue box.

6. Turn on sufficient fluidics stations. Replace bottles of RNase free dH2O with the appropriate bottles of wash solutions and empty the waste bottle.

7. Open Fluidics dialogue box of appropriate station and run Prime wash protocol on all ports. In fluidics station LCD window 'Remove vial will appear. Lift vial and needle up, an audible click will be heard and LCD window will display Load empty vial. Replace vial into sample holder. The port should start priming.

8. Once priming has been completed the instruction to Remove vial will reappear. Lift vial and needle up as before.

9. Proceed to desired wash and stain protocol.


C.1 Test 3 GeneChip wash and stain

05.03.02

Things to note:

1. The SAPE (streptavidin-phycoerythrin) is light sensitive. Do not expose it to light more than necessary, store in the dark at 4oC. For steps (3) and (4) prepare a master mix in an amber microfuge tube or a foil wrapped tube. Before addition, pulse-vortex for 30 seconds all reagents except for goat IgG and biotinylated antibody, these should me mixed by tapping thoroughly. Mix master mix thoroughly by tapping tube, spin briefly as necessary. Store aliquots on ice in a covered ice-tub.

2. When loading GeneChip into scanner, step (9), ensure that only the green light is illuminated on the scanner.


Method

1. Once hybridisation is completed, remove fragmented cRNA from array and return to original tube. Store at -80oC.

2. Fill array with 100µl of Wash Buffer A.

3. Prepare a master mix of SAPE stain solution. For each array use: 600µl 2X Stain Buffer 540µl RNase free dH2O 48µl (50mg/ml) acetylated BSA, Invitrogen 15561-020 12µl (1mg/ml) SAPE, Molecular Probes S-866 1200µl

4. Pulse-vortex for 30 seconds to mix, spin briefly as necessary. For each array aliquot 600µl into two amber microfuge tubes, one tube for pre-antibody staining, and the other tube for post-antibody staining.

5. Prepare a master mix of antibody solution. For each array use: 300µl 2X Stain Buffer 266.4µl RNase free dH2O 24µl (50mg/ml) acetylated BSA, Invitrogen 15561-020 6µl (10mg/ml) normal goat IgG, S I-5256 3.6µl (0.5mg/ml) biotinylated antibody, Vector Labs 5800 3. 600.0µl

6. Pulse-vortex for 30 seconds to mix, spin briefly as necessary. For each array aliquot 600µl into an amber microfuge tube.

7. Open 'Fluidics'dialogue box of appropriate station, select experiment (ba) and micro1-v1 wash protocol. Select Run , and follow instructions in fluidic station LCD window (instructions are listed below).

8. 'Eject washblock' move lever to eject position, insert array, close washblock and move lever back to engage position.


'Remove vial' remove microfuge tube from sample holder.

'Load 1st stain' place microfuge tube containing antibody solution into sample holder. The word Draining should appear.

9. When stain is complete, LCD window displays 'remove vial.' Remove array and re-engage washblock.

10. Check array window for bubbles. If bubbles are present manually remove by filling with Buffer A.

11. Keep the stained array covered with foil before scanning. Make sure that the window is clean and free from dust, if necessary wipe with lens cleaning cloth.

12. Open 'Scanner' dialogue box, select experiment (ba), and then ' Start' . When instructed, load array into scanner. When scan is complete remove array from scanner.

13. Eject washblock, insert array, close washblock and move lever back to engage position. Follow instructions in fluidic station LCD window (instructions are listed below

14. 'Remove vial' remove microfuge tube from sample holder.

'Load second stain' place microfuge tube containing antibody solution into sample holder. 'Draining' should appear.

'Remove vial'

'Load third stain' place microfuge tube containing SAPE solution into sample holder.

'Remove vial'

'Load empty tube' load a clean microfuge tube into sample holder.

15. When wash is complete, LCD window displays 'Eject cartridge' . Remove array and check array window for bubbles. If bubbles are present replace array in washblock and re-engage, the array should be refilled with Buffer A.

16. Remove array, re-engage washblock and replace microfuge tube containing stain with clean microfuge tube. Washing needle should appear. The fluidics will perform a wash procedure, once completed Remove vial will appear in LCD window, once the needle has been lifted up, it will be replaced with the message 'Staining done' .

17. Check array window for bubbles. If bubbles are present manually remove by filling with Buffer A.

18. Keep the stained array covered with foil before scanning. Make sure that the window is clean and free from dust, if necessary wipe with lens cleaning cloth.

19. Open scanner dialogue box, select experiment (aa), and then 'start' . When instructed, load array into scanner. When scan is complete remove array from scanner.

20. Once all GeneChips are scanned proceed to Shutdown of GeneChip station.


C.2 GeneChip wash and stain 03.09.01


Things to note:

1. The SAPE (streptavidin-phycoerythrin) is light sensitive. Do not expose it to light more than necessary, store in the dark at 4oC. For steps (3) and (4) prepare a master mix in an amber microfuge tube or a foil wrapped tube. Before addition, pulse-vortex for 30 seconds all reagents except for goat IgG and biotinylated antibody, these should be mixed by tapping thoroughly. Mix master mix thoroughly by tapping tube, spin briefly as necessary. Store aliquots on ice in a covered ice-tub.

2. When loading GeneChip into scanner, step (9), ensure that only the green light is illuminated on the scanner.


Method

1. Once hybridisation is completed, remove fragmented cRNA from array and return to the original tube. Store at -80oC.

2. Fill array with 200µl of Wash Buffer A.

3. Prepare a master mix of SAPE stain solution. For each array use: 600µl 2X Stain Buffer 540µl RNase free dH2O 48µl (50mg/ml) acetylated BSA, Invitrogen 15561-020 12µl (1mg/ml) SAPE, Molecular Probes S-866 1200µl

4. Pulse-vortex for 30 seconds to mix, spin briefly as necessary. For each array aliquot 600µl into two amber microfuge tubes, one tube for pre-antibody staining, and the other tube for post-antibody staining.

5. Prepare a master mix of antibody solution. For each array use: 300µl 2X Stain Buffer 266.4µl RNase free dH2O 24µl (50mg/ml) acetylated BSA, Invitrogen 15561-020 6µl (10mg/ml) normal goat IgG, S I-5256 3.6µl (0.5mg/ml) biotinylated antibody, Vector Labs 5800 3. 600.0µl

6. Pulse-vortex for 30 seconds to mix, spin briefly as necessary. For each array aliquot 600µl into an amber microfuge tube. 7. Open ' Fluidics' dialogue box of appropriate station, select experiment (ba) and 'EukGE-WS2v4' wash protocol. Select 'Run' , and follow instructions in fluidic station LCD window (instructions are listed below).

8. 'Eject washblock' move lever to eject position, insert array, close washblock and move lever back to engage position.

'Remove vial' remove microfuge tube from sample holder.

'Load 1st stain' place microfuge tube containing antibody solution into sample holder. The word 'Draining' should appear.

9. When stain is complete, LCD window displays 'remove vial.' Remove array and re-engage washblock.

10. Check array window for bubbles. If bubbles are present manually remove by filling with Buffer A.

11. Keep the stained array covered with foil before scanning. Make sure that the window is clean and free from dust, if necessary wipe with lens cleaning cloth.

12. Open 'Scanner' dialogue box, select experiment (ba), and then ' Start' . When instructed, load array into scanner. When scan is complete remove array from scanner.

13. Eject washblock, insert array, close washblock and move lever back to engage position. Follow instructions in fluidic station LCD window (instructions are listed below

14. 'Remove vial' remove microfuge tube from sample holder.

'Load second stain place microfuge tube containing antibody solution into sample holder. 'Draining' should appear.

'Remove vial'

'Load third stain' place microfuge tube containing SAPE solution into sample holder.

'Remove vial'

'Load empty tube' load a clean microfuge tube into sample holder.

15. When wash is complete, LCD window displays 'Eject cartridge . Remove array and check array window for bubbles. If bubbles are present replace array in washblock and re-engage, the array should be refilled with Buffer A.

16. Remove array, re-engage washblock and replace microfuge tube containing stain with clean microfuge tube. ' Washing needle' should appear. The fluidics will perform a wash procedure, once completed Remove vial will appear in LCD window, once the needle has been lifted up, it will be replaced with the message 'Staining done' .

17. Check array window for bubbles. If bubbles are present manually remove by filling with Buffer A.

18. Keep the stained array covered with foil before scanning. Make sure that the window is clean and free from dust, if necessary wipe with lens cleaning cloth.

19. Open 'scanner' dialogue box, select experiment (aa), and then 'start' . When instructed, load array into scanner. When scan is complete remove array from scanner.

20. Once all GeneChips are scanned proceed to Shutdown of GeneChip station.


D. Shutdown of GeneChip station


Method

1. Replace Wash Buffer A and B with RNase free water.

2. Open 'Fluidics' dialogue box, and run Shutdown wash protocol on all fluidic stations used. Follow instructions in LCD window. Once wash protocol has been completed, empty waste bottles and switch fluidics stations off.

3. Open 'scanner' dialogue box and turn laser off. Close Microarray Suite and put scanner into cool-down mode. Scanner can be switched off when it has cooled (this takes approximately 15 minutes and is marked by an audible sound change).

Links
All 13 experiments using protocol P-BAIR-3