Myeloid Leukemia

224 Kohlmann et al.
available databases, including the Washington University EST trace repository
and the University of California–Santa Cruz Golden-Path human genome
database (April 2001 release). In addition, an advanced understanding of probe
uniqueness and hybridization characteristics allowed an improved selection of
probes based on predicted behavior. The U133 chip design uses a multiple
linear regression model that was derived from a thermodynamic model of
nucleic acid duplex formation (6). This model predicts probe binding affinity
and linearity of signal changes in response to varying target concentrations.
The two arrays are manufactured as standard format arrays with a feature size
of 18 µm and use 11 probe pairs per sequence. The oligonucleotide length is
25-mer.
3.3.9. Microarray Washing and Staining
After hybridizing for 16 h at 45°C, the microarray is ready for washing and
staining. GeneChip probe arrays are processed by the Fluidics Station instrument,
which contains four modules, with each module processing one
microarray cartridge.
1. Use the Microarray Suite software and define an experiment for each array to be
processed (.exp extension). Perform a priming protocol to ensure that the wash
lines are full of the appropriate buffer and that the fluidics station is ready to
process a cartridge.
2. In the meantime, after 16 h of hybridization, remove the hybridization cocktail
from the probe array and fill the probe array completely with nonstringent wash
buffer (see Note 7).
3. Insert the probe array into the designated module of the fluidics station, select the
correct experiment name in the drop-down experiment list, and start the protocol
for washing and staining of expression microarrays. Standard-format microarrays
were processed using the EukGE-WS2v4 signal-amplification protocol (see
Table 7).
4. When the liquid crystal display (LCD) window indicates, place the
microcentrifuge vial containing 600 µL of the respective staining solution into
the sample holder. Verify that the metal sampling needle is in the vial with its tip
near the bottom.
5. At the end of the run, remove the probe arrays from the fluidics station modules
and check the probe array window for large bubbles or air pockets. If the probe
array has no large bubbles, it is ready to scan on the scanner. Otherwise, fill the
array manually with non-stringent wash buffer.
3.3.10. Microarray Scanning and Image Analysis
After the wash and staining protocols are complete, the probe array is
scanned using the GeneArray scanner. The laser excitation enters through the
back of the glass support and focuses at the interface of the array surface and