Myeloid Leukemia

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Diagnosis of AML1-MTG8 (ETO)-Positive AML 153 Fig. 1. Sequential qualitative reverse-transcription (RT)-polymerase chain reaction (PCR) monitoring of AML1-MTG8 (ETO) in 6 t(8;21) acute myeloid leukemia (AML) patients. Patients 1 and 2 experienced hematological relapse, whereas patients 3–6 remain in stable remission. Solid circles, RT-PCR positive; open circles, RT-PCR negative. additional therapy in an attempt to improve clinical outcome. Therefore, the value of any technique will be significantly reduced if it fails to distinguish between patients who are in stable remission and those at high risk of relapse. For this reason, qualitative protocols for the amplification of AML1-MTG8 (ETO) with high sensitivity are not suitable for MRD monitoring, as they will detect fusion transcripts even in patients in long-term remission. Moreover, protocols with a low sensitivity will not be able to predict early relapse in a large proportion of patients. Thus, the only credible approach is a sensitive quantification of the AML1-MTG8 (ETO) fusion transcripts (Fig. 1). There are
154 Tobal and Yin two strategies for the quantification of a given transcript: competitive RT-PCR and RQ-PCR. 3.4.1. Control Gene Choosing the right control gene is important for both qualitative and quantitative RT-PCR. However, a suitable control gene for qualitative RT-PCR may not necessarily be suitable for the quantitative protocol. Control genes for quantitative RT-PCR must meet two major requirements: (1) transcript levels of the control gene must not be affected by the disease in question and (2) degradation rate of the control gene must be equal to that of the gene of interest (10). Our investigations have shown that ABL is a suitable control gene for qualitative as well as quantitative RT-PCR for most AML fusion genes. 3.4.2. Competitive RT-PCR Competitive PCR is suitable for the accurate quantification of genes or their transcripts, especially when real-time equipment is not available (11). In comparison to real-time quantification, competitive PCR could be seen as laborious and time consuming. The principle of the protocol is to simultaneously amplify two targets in the same reaction using the same set of primers. The two amplicons are the target gene itself in the test sample, and another DNA fragment, the competitor, which is typically a recombinant variation of the target gene that is characterized by a deletion or an insertion to produce a differentsized PCR product (Fig. 2). The number of copies in the original stock solution of the competitor is estimated by spectrophotometry. Dilutions of this competitor are made in a range of 1–10 10 molecules/2 µL, with a dilution at every order of magnitude on a logarithmic scale. The lane in which the competitor and target PCR products are present at equal amounts (the equivalence point) indicates the number of copies in the test sample. 3.4.3. Real-Time Quantitative RT-PCR Unlike qualitative PCR, in RQ-PCR a fluorogenic probe is positioned and hybridized between the forward and reverse primers. The probe is labeled on the 5� with a fluorogenic reporter and on the 3� with a quencher. During the extension phase of the PCR cycle, the 5�→3� exonuclease activity of Taq polymerase cleaves the hybridized probe, resulting in the release of the fluorogenic reporter (Fig. 3). This causes an increase in the fluorescence emission of the reporter dye that is proportional to the amount of PCR product accumulated. Under appropriate conditions, this increase in fluorescence signal is also proportional to the amount of template used. The level of normalized reporter signal (∆Rn) increases during PCR as the target is amplified, until the reaction reaches a plateau. At the end of PCR amplification, real-time data analysis is performed.
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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
Page 116 and 117: Detection of BCR-ABL Mutations 103
Page 118 and 119: Detection of BCR-ABL Mutations 105
Page 120 and 121: Deletion of Derivative Chromosome 9
Page 128 and 129: Diagnosis of PML-RARA-Positive APL
Page 140 and 141: Duplexed QZyme RT-PCR for APL Analy
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Page 176 and 177: Diagnosis of CBFB-MYH11-Positive AM
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
Page 206 and 207: FLT3 Mutations in AML 193 Table 2 R
Page 208 and 209: FLT3 Mutations in AML 195 Fig. 2. D
Page 210 and 211: FLT3 Mutations in AML 197 10. Kiyoi
Page 212 and 213: WT1 Expression in AML and MDS 199 1
Page 214 and 215: WT1 Expression in AML and MDS 201 T
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WT1 Expression in AML and MDS 203 1
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WT1 Expression in AML and MDS 205 T
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WT1 Expression in AML and MDS 207 F
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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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