Myeloid Leukemia

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Diagnosis of AML1-MTG8 (ETO)-Positive AML 159 Fig. 6, Analysis of real-time quantitative reverse-transcription polymerase chain reactioin assay sensitivity/reliability. Levels of fusion transcripts in PB3 and BM3 samples are unreliable, as the levels of assay sensitivity/reliability are higher than those for the fusion transcript levels. Based on the principles discussed above, two criteria for a good assay sensitivity/reliability level must be achieved for the quantification to be considered reliable. Firstly, the assay’s sensitivity/reliability level must be
160 Tobal and Yin 4. Notes 1. Some samples may contain cellular clumps; if this is the case, syringing the cells/ solution D mix (after Subheading 3.1., step 2) with an approx 21-gauge needle could help to disperse the cells and aid cellular lysis. 2. Genomic DNA contamination may be precipitated with the RNA. High levels of genomic DNA contamination could therefore affect RT-PCR results. A second round of extraction (repeat the protocol previously described) could help in eliminating or significantly reducing the level of genomic DNA contamination. 3. It is necessary to perform all preparation in a sterile cabinet to avoid cross-contamination. 4. M-MLV reverse transcriptase is a thermolabile enzyme and should be kept at all times on ice while in use, then stored at –20°C. 5. The performance of positive and negative control RT reactions is necessary for accurate analysis. 6. Although a shorter RT reaction could be performed, this tends to produce lower levels of cDNA. 7. It is necessary to perform all preparation in a sterile cabinet to avoid cross-contamination. 8. Negative RT-PCR results for the control gene (ABL) indicate a lack of goodquality amplifiable RNA or cDNA. If this is the case, re-extraction of the RNA and the performance of a new RT reaction may help. 9. Positive RT-PCR results in negative control reactions indicate cross-contamination, and the analysis should be repeated. 10. It is necessary to perform all preparation in a sterile cabinet to avoid cross-contamination. 11. Once the cDNA is prepared, the volume is then diluted to 50 µL with water. Using 5 µL of cDNA in the RQ-PCR reaction reduces variation in the level quantified due to sample handling. 12. Performing a standard curve quantification is necessary with all new sets of primers and probes. 13. A low level of ABL indicates a low amount of amplifiable RNA/cDNA. Re-extraction of RNA may help in improving the results. References 1. Raimondi, S. C., Kalwinsky, D. K., Hayashi, Y., Behm, F. G., Mirro, J. Jr., and Williams, D. L. (1989) Cytogenetics of childhood acute nonlymphocytic leukemia. Cancer Genet. Cytogenet. 40, 13–27. 2. Rowley, J. D. (1990) Recurring chromosome abnormalities in leukemia and lymphoma. Semin. Hematol. 27, 122–136. 3. Erickson, P., Gao, J., Chang, K. S., et al. (1992) Identification of breakpoints in t(8;21) acute myelogenous leukemia and isolation of a fusion transcript, AML1/ ETO, with similarity to Drosophila segmentation gene, runt. Blood 80, 1825– 1831.
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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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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
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Page 212 and 213: WT1 Expression in AML and MDS 199 1
Page 214 and 215: WT1 Expression in AML and MDS 201 T
Page 216 and 217: WT1 Expression in AML and MDS 203 1
Page 218 and 219: WT1 Expression in AML and MDS 205 T
Page 220 and 221: WT1 Expression in AML and MDS 207 F
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WT1 Expression in AML and MDS 209 2
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WT1 Expression in AML and MDS 211 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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