Myeloid Leukemia

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Duplexed QZyme RT-PCR for APL Analysis 139 tial to accurately quantitate PML-RARα transcript levels (see Note 18). This also provides defined acceptance/rejection criteria from which to minimize false-negative results (see Table 3). This feature of the duplexed quantitative assay provides a distinct advantage over qualitative methods. 1. Quantitative data are expressed as an RDC, which equals the ratio of copies of disease transcripts (expressed as the number of copies of PML-RARα in 50 ng of patient RNA) to control transcripts (expressed as pg equivalents of BCR in 50 ng of patient RNA). Calculate RDC with the following equation (see Note 8): RDC at diagnosis = PML-RARα copy# (per 50 ng of patient RNA)/BCR pg equivalents (per 50 ng of patient RNA) 2. Samples are rejected (no result) or accepted (and subsequently classified) according to estimates for both the BCR and PML-RARα. Once analyzed, patient data are classified according to one of four categories: (1) no result, (2) negative (not detectable), (3) detectable, but not quantifiable, or (4) quantifiable (see Table 3). 3.6. Validation of QZyme Assays In order to produce data that are quantitative, accurate, specific, and reproducible, it is important for each laboratory to test the critical parameters of the assay on the equipment in their laboratory. This will also permit inter-laboratory standardization of assays for multi-center clinical trials. The critical parameters to test for real-time quantitative PCR are (1) limit of detection, (2) limit of quantitation, and (3) reproducibility (intra- and inter-assay variation). In this subheading, a brief description of how to test these parameters is provided, and a summary of values for the assays used to validate this method is presented in Table 4. 3.6.1. Limit of Detection (LOD) LOD refers to the lowest level of an analyte (e.g., PML-RARa transcript) that can be detected above background (see Note 19). Background signal is determined by analyzing 10 replicates of negative controls, i.e., a sample without the gene transcript of interest. A suitable negative control for PML-RARα is RNA from Meg-01 (a cell line that does not express the PML-RARα transcript). As BCR is constitutively expressed, an RNA-containing negative control does not exist. Water is used as the negative control for BCR. The LOD for the duplexed QZyme PCR PML-RARα-BCR assay is determined using the following protocol: 1. Set up duplex QZyme PCR PML-RARα-BCR reactions as outlined under Subheading 3.4. 2. At Subheading 3.4., step 3, add 5 µL of 100 ng/µL Meg-01 total RNA to 10 separate reaction tubes, and 5 µL of water to a further 10 reaction tubes.
140 Mokany et al. Table 4 Summary of Values for Each Assay QZyme assay L-type / V-type S-type BCR Limit of detection (threshold 40 40 40 (see cycle [Ct]) Subheading 3.6.1., step 6) Upper limit of quantitation 5 × 10 5 (
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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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Page 102 and 103: Quantitative PCR for Monitoring CML
Page 104 and 105: Quantitative PCR for Monitoring CML
Page 106 and 107: Detection of BCR-ABL Mutations 93 5
Page 108 and 109: Detection of BCR-ABL Mutations 95 F
Page 110 and 111: Detection of BCR-ABL Mutations 97 2
Page 112 and 113: Detection of BCR-ABL Mutations 99 o
Page 114 and 115: Detection of BCR-ABL Mutations 101
Page 120 and 121: Deletion of Derivative Chromosome 9
Page 128 and 129: Diagnosis of PML-RARA-Positive APL
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Page 176 and 177: Diagnosis of CBFB-MYH11-Positive AM
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FLT3 Mutations in AML 189 12 FLT3 M
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FLT3 Mutations in AML 191 3.1.1. DN
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FLT3 Mutations in AML 193 Table 2 R
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FLT3 Mutations in AML 195 Fig. 2. D
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FLT3 Mutations in AML 197 10. Kiyoi
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WT1 Expression in AML and MDS 199 1
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WT1 Expression in AML and MDS 201 T
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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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