Myeloid Leukemia

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Duplexed QZyme RT-PCR for APL Analysis 133 3.1. Calibration Curves for PML-RARα Calibration curves for quantifying either PML-RARα L-type/V-type transcripts (see Note 5), or PML-RARα S-type transcripts, are constructed using the appropriate PML-RARα plasmids (see Subheading 2.1., steps 1 and 2) diluted in a background of total RNA from Meg-01 (see Subheading 2.1., step 3). The following instructions will refer to a generic “plasmid,” applicable to the plasmid suitable for the breakpoint occurring in the patient cohort being tested. Calibrator 1 is stored frozen in single-use aliquots, from which all other calibrators are made (by serial dilution as discussed later) on the day of the experiment (see Note 6). It is recommended that concentrations of total RNA
134 Mokany et al. 3.2. Calibration Curves for BCR Calibration curves for quantifying BCR transcripts are constructed by diluting Meg-01 total RNA in Nuclease-free water. 1. Make up multiple aliquots of 120 µL of 100 ng/µL Meg-01 total RNA and store frozen as Calibrator 1 for BCR curve. Calibrators 2–5 are made by performing fourfold serial dilutions of Calibrator 1 as follows: a. For Calibrator 2 (25,000 pg Meg-01 total RNA/µL): add 5 µL of Calibrator 1 to 15 µL of nuclease-free water. b. For Calibrator 3 (6250 pg Meg-01 total RNA/µL): add 5 µL of Calibrator 2 to 15 µL of nuclease-free water. c. For Calibrator 4 (1563 pg Meg-01 total RNA/µL): add 5 µL of Calibrator 3 to 15 µL of nuclease-free water. d. For Calibrator 5 (391 pg Meg-01 total RNA/µL): add 5 µL of Calibrator 4 to 15 µL of nuclease-free water. 3.3. Extraction and Quantitation of Total RNA All RNA extractions are performed in a biohazard-protection hood (see Note 7). Two different methods are described to extract total RNA: (1) from patient samples, and (2) from cultured cells. 3.3.1. Extraction of RNA From Patient Specimens 1. For the extraction of RNA from patient specimens, use TRIzol reagent (Life Technologies) according to the manufacturer’s recommendations. Be sure to work quickly and keep patient samples ice-cold to minimize degradation. 2. To measure concentration of total RNA, centrifuge at 10,000g for 15 s and return to ice. Measure the absorbance of a sample of the concentrated stock at 260 nm on the ultraviolet spectrophotometer (following manufacturer’s instructions for use). Calculate the concentration of the RNA solution using the following formula: Concentration (µg/mL) = 40 × A 260 × dilution factor 3. Where possible, dilute diagnostic RNA samples (pretreatment) to 10 ng/µL and follow-up samples (posttreatment) to 100 ng/µL in nuclease-free water. Aliquot at least two 12-µL single-use samples (allows 5-µL duplicates to be analyzed per experiment and repeated) to avoid freeze thawing of samples (see Note 8). 4. Keep concentrated stocks of total RNA on dry ice at all times and return to –80°C storage when completed. Smaller aliquots of the concentrated stock can be made where possible to avoid freeze thawing of the entire sample for further analysis of other transcripts of interest. 3.3.2. Extraction of Total RNA From Cultured Cells For extraction of total RNA from cultured cells, follow the instructions of “QIAamp RNA mini protocol” (“QIAamp RNA blood mini handbook” from
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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 96 and 97: 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
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Page 128 and 129: Diagnosis of PML-RARA-Positive APL
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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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