Myeloid Leukemia

Classification of AML by DNA-Oligonucleotide Microarrays 215
3. Carefully remove the cell circlet with a 10-mL pipet and transfer it into a new
tube.
4. Add 10 mL of PBS.
5. Centrifuge 10 min at 370g (with brake).
6. Count the cells.
7. Place 5 × 10 6 cells into a 1.5-mL reaction tube and centrifuge 5 min at 2000 rpm
in a standard centrifuge and remove supernatant.
8. Lyse with 300 µL guanidine isothiocyanate buffer (RLT buffer, Qiagen) by
pipetting five times up and down. Subsequently, the stabilized lysates can be
stored at –80°C until preparation for microarray analyses.
3.3. Microarray Target Preparation
Figure 1 outlines the major steps of the procedure for gene expression profiling
analyses (as recommended by Affymetrix, Inc., Santa Clara, CA).
3.3.1. Isolation of Total RNA
Isolation of total RNA from frozen lysates of mononuclear cells can be performed
according to the RNeasy Mini Kit protocol (Qiagen, Hilden, Germany),
including an initial homogenization step. In this protocol, a specialized highsalt
buffer system allows up to 100 µg of RNA longer than 200 bases to bind to
the RNeasy silica-gel membrane. The biological samples are first lysed and
homogenized in the presence of a highly denaturing guanidine isothiocyanate
(GITC)-containing buffer, which immediately inactivates RNases to ensure
isolation of intact RNA. Then ethanol is added to provide appropriate binding
conditions and the sample is applied to an RNeasy mini column, where the
total RNA binds to the membrane and contaminants are efficiently washed
away. High-quality RNA is subsequently eluted in 40 µL of nuclease-free water.
Up to eight individual samples can be processed in parallel. All steps of the
protocol should be performed quickly at room temperature. All centrifugation
steps are performed in a standard microcentrifuge (Eppendorf, Hamburg, Germany).
RPE wash buffer is supplied as a concentrate. Before using it for the
first time, 4 vol of absolute ethanol have to be added to obtain a working solution.
A 70% ethanol solution is prepared in 2.0-mL reaction tubes using absolute
ethanol and nuclease-free water.
1. Thaw frozen cell lysates of individual patient samples (stored at –80°C) on ice.
Then incubate samples for 4 min at 45°C.
2. To homogenize the sample, pipet the lysate directly onto a QIAshredder spin
column, placed in a 2-mL collection tube, and centrifuge for 2 min at maximum
speed (see Note 2).
3. Add one volume (usually 300 µL) of 70% ethanol to the homogenized lysate in
the collection tube and mix well by pipetting. Do not centrifuge. Apply the