4 protocols
AccessionNameType
Affymetrix.com:Protocol:Percentile
feature_extraction
Title: CEL Analysis. Description:
P-BAIR-1
nucleic_acid_extraction
TOTAL RNA EXTRACTION FROM TISSUES

Homogenize tissue samples in mortar with excess amount of liquid nitrogen. Measure the weight of homogenized samples.

Transfer 50-100 mg of tissues in 50ml tube having 3 ml of TRIzol reagent (Invitrogen). Homogenize the samples using Tekmars TISSUMIZER or Polytron or equivalent. Always keep the tube in ice so as to prevent the overheating.

Optional Step: Additional step may be required for samples with high content of proteins, fat, polysaccharides or extra cellular materials such as muscles, fat tissue and tuberous parts of the plants. Following homogenisation, remove insoluble materials by centrifugation at 12,000 g for 10 minutes at 2-4 .C. The resulting pellet contains insoluble materials and the supernatant contains RNA. In samples from fat tissue, an excess of fat collects as a top layer, which should be removed. Transfer the clear homogenate solution in a fresh tube.

Phase Separation: Incubate the sample at 15 to 30 .C (RT) for 5 minutes to permit for the complete dissociation of nucleoprotein complexes. Add 0.2 ml of chloroform (high grade) per 1 ml of TRIzol reagent. Shake tubes vigorously for 15 seconds and incubate them at 15 to 30 .C (RT) for 2 to 3 minutes. Centrifuge the samples at 12000 g for 15 mints at 2 to 4.C. Following centrifugation; the mixture separates into a lower red, phenol-chloroform phase, an interphase, and a colourless upper aqueous phase. RNA remains exclusively in the aqueous phase. The volume of the aqueous phase is about 60% of the volume of TRIzol used for homogenisation.

RNA Precipitation: Transfer the aqueous phase to a fresh tube and save the organic phase if isolation of DNA or protein is desired. Precipitate RNA from the aqueous phase by mixing with isopropyl alcohol. Use 0.5 ml of Isopropyl alcohol for every 1ml of TRIzol used. Incubate samples at 15 to 30 .C for 10 minutes and centrifuge at 12000g for 10 minutes at 2-4.C.

Remove the supernatant carefully without disturbing the pellet. Wash the RNA pellet once with 75% alcohol, adding at least 1ml of 75% ethanol for every 1 ml of TRIzol used. Mix the sample by vortexing slowly and centrifuge at 7500 g for 5 minutes at 2-4.C.

Remove the supernatant carefully without disturbing the pellet. Dry the RNA pellet in air for 10 minutes. Dissolve the RNA pellet in 100 ul of RNase free water. Pipette out few times and transfer in eppendorf tube and store in 70.C.

Total RNA is further cleaned up by using RNeasy mini kit (Qiagen) Cat No:74104

Step 1: 350ul of RLT buffer is required for each total RNA sample. Take the aliquot required in a tube (If you are going to clean 10 samples, calculate for 12 samples and take 12*350= 4.2 ml) and add 10ul of B-Mercaptoethanol (Sigma-Molecular Biology Grade) per 1ml of RLT buffer (42ul for 4.2 ml of RLT buffer).
Step 2: Add 350 of RLT Buffer (B-Mercaptoethanol added) to 100ul of total RNA sample (if the volume is less make up to 100ul) and mix thoroughly. Add 250ul of 100% ethanol. Mix well by pipetting. Do not centrifuge.
Step 3: Apply sample (Should be around 700ul) to an RNeasy mini spin column, sitting in a collection tube. Centrifuge in cold for 20 sec at 8000g. You can apply the flow through in the column for twice to improve the yield. Discard the flow through as well the tube after repeating step 3 twice. Keep the column in a fresh 2ml tube.
Step 4: Apply 500ul of RPE buffer (please make sure that alcohol is added in RPE buffer) on to the column. Centrifuge for 15 sec at 8000g. Discard the flow through.
Step 5: Apply 500ul of RPE buffer and centrifuge for 2 min at max speed. Discard the flow through. Spin again at max speed for a minute. Transfer the column in a fresh RNase free collection tube.
Step 6: Pipette 30ul of RNase free water on to the column. Heat the sample at 55.C for not more than 2 minutes (to get maximum yield). Centrifuge at 8000g for a minute. Repeat step 6 once more to get 60ul of clean total RNA and proceed with QC and OD measurements. It is always to better keep the stock in two-three aliquots (to avoid freeze-thaw cycle) before freezing them in dry ice and transfer them to -80.

P-BAIR-2
labeling
cRNA preparation using phenol
These protocols are based on those provided by Affymetrix and include modifications from the Whitehead Institute for Biomedical Research, MIT, Boston and the CSC/IC Microarray Centre.

A. First strand cDNA synthesis 5.11.01
Things to note:

1.It is very important to start with good quality, clean, total RNA. Acceptable ratios for the absorbance of the total RNA at 260nm and 280nm are between 1.9 and 2.1. Ensure that the total RNA is diluted, so that the absorbances measured are in the linear range for the spectrophotometer. This is often between 0.1 and 0.4 absorbance units.
2.For step (1) we recommend starting with 10µg total RNA per reaction tube. Do not start with less than 10µg total RNA per reaction. Carry out two reactions for each sample if you require more than 40µg of cRNA.
3.Steps (3) and (4) are key to the preparation of cDNA. Step (3) breaks any secondary structures of the RNA while step (4) inhibits the reoccurrence of secondary structures.
4.Make a 'master mix' for step (5). Mix thoroughly and spin briefly, as necessary.
5.Use RNase free microfuge tubes and pipette tips throughout.

Method
1. Place the following in a microfuge tube:
-µl (10µg) total RNA in nuclease free treated dH2O
-µl (100 pmol) T7-(T)24 primer
(GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24)
1µl Poly A+ Controls
-µl nuclease free dH2O
11 µl final volume
2.Mix by pipetting, spin briefly as necessary.
3.Incubate at 65-70oC for 10 minutes.
4.Place tubes on ice.
5.Prepare a master mix on ice. The quantities are for each reaction/tube:
4µl (5x) First Strand Buffer (thaw at 37oC, put on ice), Invitrogen Y02321
2µl (0.1 M) DTT, Invitrogen Y00147
1µl (10mM) dNTPs, Invitrogen 18427-013
7µl
Mix by pipetting, spin briefly as necessary.Add 7µl of master mix to each reaction tube.
6.Incubate at 42 oC for 2 minutes.
7.Add 2µl Superscript II reverse transcriptase (400 U total), Invitrogen 18064-014.
8.Tap tube to mix, spin briefly as necessary.
9.Incubate at 42oC for 1 hour in a waterbath.
10.Place tube on ice and proceed to 'Second strand cDNA synthesis', or transfer tube to dry ice, allow reaction to freeze and store at -80oC.

B. Second strand cDNA synthesis 12.02.01.

Things to note:
1.Check the units/µl for the RNase H, it is important that exactly 2U are used per reaction; you may have to adjust volumes accordingly.
2.Make a 'master mix' for step (2). Mix thoroughly because the enzymes are in glycerol. Spin briefly, as necessary.
3.For step (4), incubate either in a water bath in the cold room or in a refrigerated block with a lid, to reduce condensation.
4.For step (5), store at -80oC only if you cannot go to the 'Clean-up of double stranded cDNA' step immediately after the second strand cDNA synthesis is completed.

Method
1.Place all reagents and first strand reaction tubes on ice. Assemble master mix on ice.
2.Prepare a master mix in a new microfuge tube. For each reaction use:
-µl nuclease free treated dH2O
30µl (5x) Second Strand Buffer, Invitrogen 10812-014
3µl (10mM) dNTPs, Invitrogen 18427-013
1µl DNA Ligase (10 Units), Invitrogen 18052-019
4µl DNA Pol I (40 Units), Invitrogen 18010-025
-µl RNase H (2 Units), Invitrogen 18021-071
130µl
Mix by vortexing, spin briefly as necessary. Add 130µl of the master mix to each of the first strand reaction tubes.
3.Mix by pipetting, spin briefly as necessary.
4.Incubate at 16oC for 2 hours. Use a water bath in a cold room or a refrigerated block with lid, if possible. This reduces condensation.
5.Place tube on ice and proceed to 'Clean-up of double stranded cDNA', or transfer tube to dry ice, freeze and store at -80oC.

C. Clean-up of double stranded cDNA 25.09.02.

Things to note:
1. Double stranded cRNA clean up is carried out using the GeneChipý Sample Cleanup Module, Affymetrix 900371. Prepare reagents following the instructions in the kit.
2. Perform the clean up in a 1.5 or 2ml microfuge tube.
3. Ensure ethanol has been added to cDNA Wash Buffer before use.
4. After the clean up, ALL of the double stranded cDNA is used for the in vitro transcription reaction. Do not attempt to quantify the products of the reaction.

Method
1. Add 600µl cDNA Binding Buffer to the cDNA reaction. Mix by pipetting.
2. Check that the colour of the mixture is yellow (If the colour is orange or violet, add 10µl of 3 M sodium acetate, pH 5.0 and mix).
3. Apply 500µl of the sample to the cDNA Cleanup Spin Column.
4. Centrifuge at >=8 000g (>= 10 000rpm) for 1 minute.
5. Empty the collection tube.
6. Reload the spin column with the remaining mixture.
7. Centrifuge at >=8,000g (>= 10,000rpm) for 1 minute.
8. Transfer the spin column into a new 2ml Collection tube.
9. Pipette 750µl cDNA Wash Buffer onto the spin column.
10.Centrifuge at >=8 000g (>= 10 000rpm) for 1 minute.
11.Empty the collection tube.
12.Open the cap of the spin column and centrifuge at maximum speed for 5 minutes, to completely dry the membrane.
13.Transfer the spin column to a fresh 1.5ml collection tube.
14.Elute by placing 14µl of cDNA Elution Buffer directly onto the middle of the spin column membrane.
15.Incubate at room temperature for 1 minute.
16.Centrifuge at maximum speed for 1 minute.
17.Place tube on dry ice to freeze and then store at-80oC or proceed directly to the in vitro transcription reaction.

D. Preparation of cRNA 07.03.03

Things to note:
1.Once reagents have thawed, keep them at room temperature until incubation at 37oC to reduce precipitation of DTT.
2.The incubation should be carried out in an incubator or warm-room, to reduce condensation on the inside of the lid.
3.The reagents used for in vitro transcription are from the Bioarray High Yield RNA Transcript Labelling Kit, Enzo 900182.

Method:
1.Thaw all reagents and double stranded cDNA at room temperature.
2.Prepare a master mix of the IVT reagents at room temperature. The quantities are for each reaction/tube:
12µl double stranded cDNA from previous method.
10µl nuclease free dH20
4µl (10x) HY Reaction Buffer
4µl Biotin-labelled ribonucleotides
4µl DTT
4µl RNase Inhibitor Mix
2µl T7 RNA Polymerase
40µl
3.Mix by pipetting, spin briefly as necessary.
4.Incubate at 37oC for 5 hours. Gently mix the reaction every hour by tapping tube, spin briefly as necessary.
5.Proceed to 'Clean-up of cRNA', or freeze by placing tube on dry ice and store at -80oC.

E. Clean-up of cRNA 07.03.03.

Things to note:
1.Removal of unincorporated biotinylated ribonucleotides is very important
obtaining a good quality scan.
2. cRNA clean up is carried out using the GeneChip Sample Cleanup Module,Affymetrix 900371. Prepare reagents following the instructions in the kit.
3. Ensure ethanol has been added to IVT cRNA Wash Buffer before use.
4. Be careful not to touch the tip of the spin column at any time.
5. Carry out centrifugation at >= 8,000g (>= 10,000rpm).

Method:
1.Add to the in vitro transcription reaction tubes:
60µl nuclease free dH2O
350µl IVT cRNA Binding Buffer
2.Mix thoroughly by pipetting.
3.Add 250µl (100%) ethanol. Mix by pipetting. Do not spin.
4.Apply sample to an IVT cRNA Cleanup Spin Column.
5.Spin at >= 8,000g (>= 10,000rpm) for 15 seconds.
6.Transfer the spin column to a fresh 2ml collection tube.
7.Add 500µl IVT cRNA Wash Buffer onto the spin column.
8.Spin at >= 8,000g (>= 10,000rpm) for 15 seconds.
9.Empty the collection tube.
10.Add 500µl (80%) ethanol onto the spin column.
11.Spin at >= 8,000g (>= 10,000rpm) for 15 seconds.
12.Empty the collection tube.
13.Open the cap of the spin column and centrifuge at maximum speed for 5 minutes to completely dry the membrane.
14.Transfer spin column into a new 1.5ml Collection tube.
15.Elute by placing 11µl of RNase-free water directly onto the middle of the spin column membrane.
16.Incubate at room temperature for 2 minutes.
17.Spin at maximum speed for 1 minute.
18.Add 10µl of RNase-free water directly onto the middle of the spin column membrane.
19.Incubate at room temperature for 2 minutes.
20.Spin at maximum speed for 1 minute.
21.Dilute an aliquot of the eluate with dH2O (not DEPC treated) and measure the absorbance at 260nm and 280nm. The dilution should be sufficient so that readings obtained are in the linear range for the spectrophotometer. Unless known otherwise assume this is between 0.1 and 0.4 absorbance units (try preparing a serial dilution, diluting 2µl of eluate in 48µl of dH2O and then this 50µl in 450µl of dH2O). The cRNA should be clean e.g. a ratio of the absorbances at 260nm to 280nm should be around 2.0, with an acceptable range of 1.9 to 2.1. You should have 40 to 110µg of cRNA (assuming that you started with 10µg total RNA where 1 OD260 unit is equivalent to 40µg/ml single stranded RNA). If you have a yield of less than 40µg of cRNA per reaction (per 10µg reaction of total RNA), we recommend that you do not use the cRNA, as there is likely to be a problem with sample preparation.
22.Run a sample of the total RNA and cRNA for each sample (500ng to 1µg) on a denaturing 1% agarose gel along with a marker spanning 200bp to 3kb (a DNA marker gives a rough idea of the size). The cRNA product should be a smear from about 100bp to 2kb with a brighter region from 500bp to 1kb, there is an example image for guidance at the end of this protocol. Photograph the gel, so that the markers are easy to identify and label the lanes with the sample identifier.
23.Freeze by placing tube on dry ice and store at 80 °C.
24.Bring the cRNA to the Microarray Centre on dry ice (after prior consultation with Helen Banks or Nicola Cooley to arrange a convenient time). Note that each sample should be clearly labelled on both the top and side of each tube with the date (day, month, year e.g. 05.11.01) and the sample identifier.
In order to assess the quality of the cRNA samples, prior to hybridisation on test arrays, we also require the following information, for both the cRNA samples and the total RNA from which it was generated.
(a) the raw absorbance readings
(b) the ratio of the absorbances at 260nm and 280nm.
(c) the cRNA yield from 10µg of total RNA.
(d) an original photograph of the gel, with the markers and samples labelled.
P-BAIR-3
hybridization
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).