Myeloid Leukemia

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Classification of AML by DNA-Oligonucleotide Microarrays 235 Fig. 6. Classification task. The support vector machine separates a given set of binary labeled training data with a hyperplane that is maximally distant from them (maximal margin). The middle black line is the decision surface defining the borderline between the area of prediction of type I samples (squares) and type II samples (circles). The outer lines precisely meet the constraint. Support vectors marked as critical for the classification task are the points that lie closest to the separating hyperplane. 3.4.6. Functional Gene Annotation: NetAffx Database The NetAffx database can be used to functionally annotate the probe sets represented on the microarrays. The NetAffx Analysis Center (www.affymetrix.com/analysis/) is an integrated, freely available online resource created by Affymetrix (21). This Web-based application enables researchers to correlate results from experiments with array design and annotation information. It is a dynamic tool and provides access to underlying array content and the design of GeneChip probe arrays, including probe sequences and extensive gene annotations from both Affymetrix and the public domain. It allows the user to search array contents for sequences of interest, review gene and protein characterizations for represented probe sets, and sort transcripts by functional group, metabolic pathway, and disease association. For each cataloged Affymetrix GeneChip microarray, an anchoring databank summarizes all the annotations for the probe sets. The information provided for each probe set falls into two categories: sequence annotations and static information. Sequence annotations refer to the information about the representative sequence for a probe set, including functional annotations for gene title, gene
236 Kohlmann et al. symbol, and cytogenetic bands. The static information for each probe set details the probe sequences, accession numbers, and textual description, and describes what the probes were designed to interrogate. The static probe set data are also depicted graphically. 3.4.7. Software Software packages from Affymetrix (www.affymetrix.com/support/) are used for principal data acquisition (MAS5), storage (MicroDB), and analysis (DMT). Individual gene expression profiles can also be prepared as Microsoft Excel tables. The following packages can be applied for identification of differentially expressed genes and classification: SAM (Stanford University; www-stat.stanford.edu/~tibs/SAM/index.html) Bioconductor (open source; www.bioconductor.org) q-value (University of Washington; faculty.washington.edu/~jstorey/qvalue/) LIBSVM (National Taiwan University; www.scie.ntu.edu.tw/~cjiln/libsvm/). SAM is available as a Microsoft Excel Add-in. Bioconductor is an open source and open development software project for the analysis and comprehension of genomic data (22). Bioconductor packages provide statistical and graphical methodologies for analyzing genomic data. LIBSVM (Version 2.6) is a software solution for SVM-based classification. The q-value software takes a list of p-values resultling from the simultaneous testing of many hypotheses and estimates their q-values (13). In addition, further third-party software packages can be used for statistical analyses and data visualization: SPSS (SPSS Inc.; www.spss.com/) Pathways Analysis (Ingenuity Systems; www. Ingenuity.com) GeneMaths (Applied Maths, Inc.; www.applied-maths.com) J-Express (MolMine AS; www. molmine.com/).www.applied-maths.com 4. Notes 1. The whole sample target preparation procedure can be performed in two working days, taking the assay’s safe stopping points into account. Day 1 includes isolation of total RNA, synthesis of ds cDNA, cleanup of ds cDNA, and ethanol precipitation overnight. The IVT reaction, cRNA cleanup, quantification, and fragmentation are performed on day 2. After a hybridization cocktail has been prepared, it is either subsequently hybridized to a probe array, or can be stored at –20°C for later use. Throughout all steps, powder-free gloves should be worn. All steps to minimize the introduction of exogenous nucleases should be taken. Water used in the protocols has to be molecular-biology grade. All steps should be performed in nuclease-free 1.5-mL reaction tubes.
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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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Diagnosis of PML-RARA-Positive APL
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Duplexed QZyme RT-PCR for APL Analy
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Diagnosis of AML1-MTG8 (ETO)-Positi
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Diagnosis of CBFB-MYH11-Positive AM
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165 Table 1 Primers for CBFB-MYH11
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FIP1L1-PDGFRA in Hypereosinophilic
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Page 202 and 203: FLT3 Mutations in AML 189 12 FLT3 M
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Page 212 and 213: WT1 Expression in AML and MDS 199 1
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Page 266 and 267: Detecting JAK2 V617F Mutation in MP
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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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