Myeloid Leukemia

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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
216 Kohlmann et al. Fig. 1. Gene expression analysis overview. The gene expression profiling analysis starts with sample target preparation (see Note 1). The target is the labeled nucleic acid that is being interrogated. It is hybridized to the probes on the array. For each respective sample, double-stranded (ds) cDNA is synthesized from total RNA isolated from mononuclear cells. An in vitro transcription (IVT) reaction is then performed to produce biotin-labeled cRNA from the cDNA. After fragmentation, a hybridization cocktail is prepared, including the fragmented target, probe array controls, bovine serum albumin, and herring sperm DNA. The cocktail is hybridized to the probe array during a 16-h incubation. Immediately following hybridization, the probe array undergoes an automated washing and staining protocol on the fluidics station. After the array is scanned, the raw data are analyzed for probe signal intensities and all results are reported in tabular and graphical formats. Then the data set is prepared for detailed statistical analyses.
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M E T H O D S I N M O L E C U L A R
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M E T H O D S I N M O L E C U L A R
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© 2006 Humana Press Inc. 999 River
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vi Preface comprehensive review of
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viii Contents 11 Detection of the F
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x Contributors D. GARY GILLILAND
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RNA and DNA From Leukocytes 1 1 Iso
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RNA and DNA From Leukocytes 3 mine
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RNA and DNA From Leukocytes 5 late
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RNA and DNA From Leukocytes 7 9. Us
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RNA and DNA From Leukocytes 9 16. C
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RNA and DNA From Leukocytes 11 3. I
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Cytogenetic and FISH Techniques 13
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Real-Time RT-PCR Strategies 27 3 Ov
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Real-Time RT-PCR Strategies 29
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Real-Time RT-PCR Strategies 31 cifi
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Real-Time RT-PCR Strategies 33 2.1.
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Real-Time RT-PCR Strategies 35 Fig.
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Real-Time RT-PCR Strategies 37 2.1.
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Real-Time RT-PCR Strategies 39 the
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Real-Time RT-PCR Strategies 43 duri
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MyiQ single color 400 nm 585 nm 96
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Real-Time RT-PCR Strategies 47 side
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Table 2 Real-Time Quantitative Poly
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Real-Time RT-PCR Strategies 51 AML-
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Real-Time RT-PCR Strategies 53 the
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Real-Time RT-PCR Strategies 55 plas
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Real-Time RT-PCR Strategies 57 betw
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Real-Time RT-PCR Strategies 59 Ct C
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Real-Time RT-PCR Strategies 63 asse
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Real-Time RT-PCR Strategies 65 21.
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Real-Time RT-PCR Strategies 67 50.
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Quantitative PCR for Monitoring CML
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Detection of BCR-ABL Mutations 93 5
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Detection of BCR-ABL Mutations 95 F
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Detection of BCR-ABL Mutations 97 2
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Detection of BCR-ABL Mutations 99 o
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Detection of BCR-ABL Mutations 101
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Deletion of Derivative Chromosome 9
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Diagnosis of PML-RARA-Positive APL
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Duplexed QZyme RT-PCR for APL Analy
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Diagnosis of AML1-MTG8 (ETO)-Positi
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Diagnosis of CBFB-MYH11-Positive AM
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Page 190 and 191: FIP1L1-PDGFRA in Hypereosinophilic
Page 202 and 203: FLT3 Mutations in AML 189 12 FLT3 M
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Page 212 and 213: WT1 Expression in AML and MDS 199 1
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Page 220 and 221: WT1 Expression in AML and MDS 207 F
Page 226 and 227: Classification of AML by DNA-Oligon
Page 246 and 247: 233 Classification of AML by DNA-Ol
Page 254 and 255: Classification of AML by Monoclonal
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Page 266 and 267: Detecting JAK2 V617F Mutation in MP
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PRV-1 Overexpression in PV and ET 2
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Chimerism Analysis 275 18 Chimerism
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Chimerism Analysis 277 1.2. Detecti
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Chimerism Analysis 279 3. Extractio
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Chimerism Analysis 281 14. 310 Gene
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Chimerism Analysis 283 12. Upon com
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Chimerism Analysis 285 Fig. 2. Calc
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Chimerism Analysis 287 4. Allow the
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Chimerism Analysis 289 capillary el
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Chimerism Analysis 291 and recipien
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Chimerism Analysis 293 Table 2 Comm
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Chimerism Analysis 295 12. Maas, F.
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298 Index management, 128 AML, see
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300 Index and AML, 213, 236, 238, 2
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302 Index chimerism analysis in sex
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304 Index minimal residual disease
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306 Index Stem cell transplantation
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Myeloid Leukemia

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