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cartridge with 250 μL of the non-stringent wash buffer. The array is now ready for washing and staining in the fluidics station. As the station can only process four arrays at a time, other arrays to be processed can be stored temporarily at 4 °C for up to 3 h. 5. The fluidics station needs to be primed to ensure the lines are filled with the appropriate buffers and is ready for running protocols. The non-stringent wash buffer should be filled in the Wash A buffer reservoir on the machine. The stringent wash buffer should be filled in the Wash B buffer reservoir. Run the Prime_450 maintenance protocol with empty microcentrifuge tubes in the stain holder positions 1, 2, and 3. 6. After priming, the fluidics station is ready to accept arrays for washing and staining. Using the proper protocol enter the sample file name, the array name, and the probe array type. Select the fluidics protocol script for processing the arrays. For our experiments, the fluidics script used was the EukGE- WS2v5_450. Follow instructions on the LCD window on the fluidics station for loading the array into the machine and for loading of the sample holders. There are three sample holders on the machine module. Place one vial containing 600.0 μL SAPE stain solution in sample holder 1. Place one vial containing 600.0 μL antibody solution in sample holder 2. Place one vial containing 600.0 μL SAPE stain solution in sample holder 3. Press down on the needle lever to snap needles into position which will start the run. 7. When the protocol is complete, the LCD window will display the message EJECT & INSPECT CARTRIDGE. Press down on the cartridge lever to the eject position and remove the array. Do not engage the washblock until the array has been inspected for the presence of bubbles or air pockets. If the array has no bubbles, it is ready for scanning. If there are bubbles present, reinsert the array back into the washblock probe array holder and engage the washblock. The array will be drained and refilled. Recheck the array for any bubbles and when none are present, continue on scanning the array. Engage all washblocks for the fluidics station to continue to complete the protocol and prime for the next wash protocol. 8. If the arrays are not scanned immediately following washing and staining, they can be stored at 4 °C, in the dark, until ready for scanning for a maximum of 24 h. 9. A shutdown protocol should be run on the fluidics station at the end of the daily session. 3.3 Microarray Scanning
The scanning of the Affymetrix GeneChip® Rat Genome 230 2.0 Arrays are automated using the Affymetrix GeneChip® Scanner 3000. The sample file created using the GCOS software (or the
