Myeloid Leukemia

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Diagnosis of CBFB-MYH11-Positive AML 167 control. Alternatively, CBFB-MYH11 plasmid constructs may be applied. To exclude false-positive and false-negative results, the dilution series should be included in each analysis. The positive control must be diluted in such a manner that at a certain dilution a conversion from positive to negative signals is observed. If no conversion from PCR positive to negative signals is observed, the dilution series may be contaminated. Consequently, false-negative results may be obtained due to failure to recognize inadequate sensitivity of the assay. To show that larger transcripts are efficiently amplified in qualitative PCR, we recommend also including a patient sample positive for CBFB-MYH11 fusion transcript types D or E (see Fig. 1). To avoid false-positive results, several control samples negative for the CBFB-MYH11 fusion should be included in each PCR run. 3.1.1. Qualitative CBFB-MYH11 PCR 1. Prepare the first-round CBFB-MYH11 PCR mix by pipetting in the following order for one PCR reaction (see Notes 2 and 3): 11.09 µL H 2O, 1.25 µL 50 mM MgCl 2, 0.4 µL dNTPs, 2.5 µL DMSO, 2.5 µL 10X PCR buffer, 1.0 µL primer cd (Table 1), 1.0 µL primer mm (Table 1), and 0.26 µL Taq DNA polymerase (total volume of 20 µL). 2. Mix the first-round CBFB-MYH11 PCR mix and add per reaction 5 µL of cDNA (50 ng) and a drop of mineral oil (see Note 4). 3. Perform the first-round CBFB-MYH11 PCR using an initial denaturing step of 5 min at 94°C, followed by 27 cycles of 1 min at 94°C (denaturation of cDNA- RNA hybrids), 1 min at 62°C (primer annealing), 1 min at 72°C (product extension), followed by a final extension step of 10 min at 72°C (see Note 5). 4. Prepare the second-round CBFB-MYH11 PCR mix by pipetting in the following order for one PCR reaction: 11.09 µL H 2O, 1.25 µL 50 mM MgCl 2, 0.4 µL dNTPs, 2.5 µL DMSO, 2.5 µL 10X PCR buffer, 1.0 µL primer cmdI (Table 1), 1.0 µL primer mmd2 (Table 1), and 0.26 µL Taq DNA polymerase (total volume 20 µL). 5. Mix the second-round CBFB-MYH11 PCR mix and add per reaction 5 µL of a five times dilution of the first-round PCR product and a drop of mineral oil. 6. Perform the second-round CBFB-MYH11 PCR using an initial denaturing step of 5 min at 94°C (denaturation of first-round PCR product), followed by 25 cycles of 1 min 94°C (denaturation), 1 min 62°C (primer annealing), 1 min 72°C (product extension), followed by a final extension step of 10 min at 72°C. 7. Judge CBFB-MYH11 PCR amplification by agarosose gel electrophoresis using a 1.5% agarose-TBE (0.5X) gel containing 35 ng/mL ethidium bromide. Products can be visualized after ultraviolet (UV) exposure of the agarose gel. Make sure to include DNA size markers that allow for proper size determination in the 250- to 1700-bp range. The PCR product lengths obtained for the various CBFB- MYH11 fusion transcripts after first- and second-round (nested) PCR, respectively, are indicated in Table 2.
168 van der Reijden and Jansen Table 2 Currently Known CBFB-MYH11 Fusion Transcripts and Amplicon Sizes Transcript type A B C D E F G H I S First PCR: cb-mm 703 916 1096 1423 1630 607 1327 1430 1318 394 Nested PCR: cmdI-mmdII 582 795 975 1302 1509 485 1205 1308 1197 272 Incidence (%) 89
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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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Page 178 and 179: 165 Table 1 Primers for CBFB-MYH11
Page 190 and 191: FIP1L1-PDGFRA in Hypereosinophilic
Page 202 and 203: FLT3 Mutations in AML 189 12 FLT3 M
Page 204 and 205: FLT3 Mutations in AML 191 3.1.1. DN
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Page 212 and 213: WT1 Expression in AML and MDS 199 1
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Classification of AML by DNA-Oligon
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233 Classification of AML by DNA-Ol
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Classification of AML by Monoclonal
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247 Classification of AML by Monocl
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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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