Contents
1 Introduction: DNA Microarrays / Ten Years Old, but no Old Hat
Falk Hertwig and Ulrike A. Nuber
1.1 How to perform a microarray experiment
2 cDNA Microarray Analysis and its Role in Toxicology / a Case Study
Alexandra N. Heinloth, Gary A. Boorman and Richard S. Paules
2.1 Introduction
2.2 Gene expression profiling reveals indicators of potential adverse effects
2.3 cDNA microarrays / curse or blessing
2.4 The future ot toxicogenomics / prediction of toxicity
Protocol 2.1: In-life study
Protocol 2.2: Gene expression analysis
Protocol 2.3: Clinical pathology
Protocol 2.4: Histopathology
Protocol 2.5: Electron microscopy
Protocol 2.6: ATP measurements
3 Gene Expression Profiling in Plants Using cDNA Microarrays
Motoaki Seki, Junko Ishida, Maiko Nakajima, Akiko Enju, Ayako Kamei, Youko Oono,
Mari Narusaka, Masakazu Satou, Tetsuya Sakurai and Kazuo Shinozaki
3.1 Introduction
3.2 Gene expression profiling methods
3.3 DNA microarrays: cDNA microarrays and oligonucleotide microarrays
3.4 cDNA clones and their application for cDNA microarray analysis
Protocol 3.1: Preparation of cDNA microarrays
Protocol 3.2: Preparation of cDNA targets
Protocol 3.3: Microarray hybridization and scanning
Protocol 3.4: Data analysis
4 Identification of Gene Expression Patterns for a Molecular Diagnosis of Kidney
Tumors
Holger Sltmann, Andreas Buneº, Markus Ruschhaupt, Wolfgang Huber, Ruprecht
Kuner, Bastian Gunawan, Laszlo Fzesi and Annemarie Poustka
4.1 Introduction
4.2 Experimental design
4.3 Molecular classification of kidney tumors
4.4 Building a classifier for kidney tumor diagnosis
4.5 Summary
Protocol 4.1: Tissue samples and RNA isolation
Protocol 4.2: Microarray experiments
Protocol 4.3: RNA labeling and hybridization
Protocol 4.4: Signal quantification and data analysis
5 Gene Expression Analysis of Differentiating Neural Progenitor Cells / a Time
Course Study
Ulf Gurok and Ulrike A. Nuber
5.1 Introduction
5.2 The experiment
5.3 Summary
Protocol 5.1: Microarray production
Protocol 5.2: Cell culture and RNA preparation
Protocol 5.3: Hybridization, washing and scanning
Protocol 5.4: Data processing
Protocol 5.5: Cluster analysis
6 A Microarray-Based Screening Method for Known and Novel SNPs
Ena Wang and Francesco M. Marincola
6.1 Introduction
6.2 High resolution SNP detection methods
6.3 High throughput methods for SNP detection
6.4 Screening methods for known and unknown SNPs
6.5 Summary
Protocol 6.1: Target preparation
7 From Gene Chips to Disease Chips / New Approach in Molecular Diagnosis of
Eye Diseases
Rando Allikmets and Jana Zernant
7.1 Introduction
7.2 APEX / arrayed primer extension
7.3 Application A / the gene array for ABCA4-associated retinal dystrophies
7.4 Application B / the 'disease array' for a genetically heterogeneous disorder (LCA)
7.5 Summary
Protocol 7.1: Template preparation
8 Multiplexed SNP Genotyping Using an Allele-Specific Primer Extension on
Microarrays
Juha Saharinen, Pekka Ellonen, Janna Saarela and Leena Peltonen
8.1 Introduction
8.2 Practical approach on microarray-based allele-specific primer extension
8.3 Data analysis / allele calling and genotype assignment
8.4 Summary
9 Profiling the Arabidopsis Transcriptome
Lars Hennig
9.1 Introduction
9.2 MIAME/Plant / documentation of the experiment
9.3 RNA extraction
9.4 Labeling
9.5 Hybridization
9.6 Washing, staining and scanning
9.7 Data pre-processing and data analysis
9.8 Useful tips
9.9 Summary
Protocol 9.1: RNA extraction
Protocol 9.2: Labeling
Protocol 9.3: Hybridization
Protocol 9.4: Washing, staining and scanning
10 Affymetrix GeneChip Analyses / the Impact of RNA Quality
Ludger Klein-Hitpass and Tarik MÜrÜy
10.1 Introduction
10.2 Aim and experimental design
10.3 Statistics of RNA and array quality parameters
10.4 Comparison of signal measures computed by different array normalization
procedures in controls and degraded samples
10.5 SAM of degraded versus control RNA
10.6 Summary
Protocol 10.1: Affymetrix GeneChip analyses
11 Molecular Karyotyping by Means of Array CGH: Linking Gene Dosage
Alterations to Disease Phenotypes
Joris Veltman and Lisenka Vissers
11.1 Introduction
11.2 Array preparation, labeling, hybridization and data analysis
11.3 Molecular karyotyping in clinical genetics
11.4 Gene identification by array CGH
11.5 Summary
Protocol 11.1: Clone preparation and array fabrication
Protocol 11.2: Array CGH procedure
Protocol 11.3: Hybridization and posthybridization procedure
12 DNA Microarrays: Analysis of Chromosomes and Their Aberrations
Heike Fiegler and Nigel Carter
12.1 Introduction
12.2 Array construction and application of genomic microarrays
12.3 Conclusion
Protocol 12.1: DOP PCR
Protocol 12.2: Aminolinking PCR
Protocol 12.3:DOP PCR amplification of flow-sorted chromosomes
Protocol 12.4: Random primed labeling of DNA for array CGH
Protocol 12.5: Array hybridization (grid size 2 \times 3 cm
13 Mapping Transcription Factor Binding Sites Using ChIP-Chip / General
Considerations
Rebecca Martone and Micheal Snyder
13.1 Introduction
13.2 Experimental approach
13.3 Experimental conditions
13.4 Data analysis
13.5 Array selection
13.6 Conclusion
14 ChIP-on-Chip: Searching For Novel Transcription Factor Targets
Esteban Ballestar and Manel Esteller
14.1 Introduction
14.2 Genomic microarrays
14.3 Performing a successful ChIP assay
14.4 Obtaining material for hybridization
14.5 Labeling and hybridizing the DNA
14.6 Validating ChIP-on-chip results
14.7 Summary
Protocol 14.1: Performing a successful ChIP assay
15 Turning Photons into Results: Principles of Fluorescent Microarray Scanning
Siobhan Picket and Damian Verdnik
15.1 Introduction
15.2 Scanning parameters
15.3 Analysis parameters
15.4 Normalization
16 Microarray Detection with Laser Scanning Device
Ralph Beneke
16.1 Introduction
16.2 CCD or PMT
16.3 Engineering of Tecan's LS series
17 Normalization Strategies for Microarray Data Analysis
Christine Steinhoff and Martin Vingron
17.1 Introduction
17.2 Experimental data
17.3 Normalization methods
17.4 Scaling methods
17.5Transformation methods
17.6 Application of normalization methods
17.7 Summary
18 Microarray Data Analysis: Differential Gene Expression
Stefanie Scheid and Rainer Spang
18.1 Introduction
18.2 Getting started
18.3 Explorative analysis
18.4 Statistical analysis
18.5 Final remarks
19 Clustering and Classification Methods for Gene Expression Data Analysis
Elizabeth Garrett-Mayer and Giovanni Parmigiani
19.1 Introduction
19.2 Clustering
19.3 Classification
19.4 Summary
20 Statistical Analysis of Microarray Time Course Data
Yu Chaun Tai and Terence P. Speed
20.1 Introduction
20.2 Design
20.3 Identifying the genes of interest
20.4 Clustering
20.5 Curve alignment
20.6 Software
20.7 Remarks
21 Array CGH Data Analysis
Yuedong Wang and Sun-Wei Guo
21.1 Introduction
21.2 Summary
21.3 Concluding remarks
22 MIAME
Robert Wagner
22.1 Introduction
22.2 The structure of MIAME
22.3 Array design description
22.4 Experiment description