Myeloid Leukemia

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Duplexed QZyme RT-PCR for APL Analysis 137 Fig. 2. Amplification plots and calibration curves for single-tube duplex assay. Examples of duplex assays for (A) PML-RARα L-type, (B) BCR control 8. Click on “thermal cycling” and program the following profile (see Note 14): 60 min at 55°C, 3 min at 95°C 10 cycles of 95°C for 15 s and 68°C for 40 s (–1°C/cycle), 40 cycles of 95°C for 15 s and 55°C for 40 s 9. Load plate, close the lid, and select “run.” 10. Save data after completion of run (see Note 15). 11. Analyze data from the ABI 7700 according to the guidelines from Applied Biosystems (Note 16). For both PML-RARα assays, set the baseline and thresholds at 3–12 and 0.2, respectively. For the BCR assays, set the baseline and threshold at 3–15 and 0.07. Examples of an amplification plot and a standard curve from the L-type duplex assay are given in Fig. 2 (see Note 17). 3.5. Relative Quantitation of Transcripts in Patient Samples To determine the correct primer set to use in QZyme RT-PCR, it is recommended that the PML-RARα isoform harbored by each patient be identified by standard qualitative RT-PCR (see Chapter 7). The single-tube QZyme RT-PCR method described in this chapter is not only able to confirm the presence of the PML-RARα transcript in each patient, but also estimate its relative abundance. The quantities of PML-RARα and BCR transcripts, amplified simultaneously in a duplex reaction, are estimated from each calibration curve (see Subheadings 3.1. and 3.2.). The relative abundance of transcripts is then calculated by normalization of PML-RARα to the control BCR transcripts, to account for variation in RNA concentration and quality, according to the equations in Subheading 3.5., step 2). The accurate assessment of RNA quality in each specimen, which can vary considerably, is essen-
Table 138 3 Mokany et al. Acceptance and Rejection Criteria of Data and Subsequent Classification of Results* PML-RARα (x copies) LOD < × < LLOQ LLOQ < × < ULOQ × < LOD (i.e., (i.e., PML-RARα (i.e., PML-RARα BCR ( y pg PML-RARα amplified, but not amplified and on equivalents) not amplified) on standard curve) standard curve) y < LLOQ No result (1) Detected but not Detected but not (i.e., not quantifiable (4) quantifiable (7) on BCR standard curve) LLOQ < y No result (2) Detected but not Quantifiable
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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 100 and 101: Quantitative PCR for Monitoring CML
Page 106 and 107: Detection of BCR-ABL Mutations 93 5
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FIP1L1-PDGFRA in Hypereosinophilic
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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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