Chapter 1
Multicenter Clinical Sample Collection for Microarray Analysis
1. Introduction
1.1. Setting up the Clinical Centers
1.2. Institutional Review Boards, HIPAA, and Compensation for Participation
1.3. Central Processing Laboratory
2. Materials
2.1. Specimen Collection Kits
2.2. RNA Extraction from PAXgene Blood Samples
2.3. RNA, DNA, and Protein Extraction from Mononuclear Cells
2.4. Plasma Separation from Whole Blood Samples (CPT)
2.5. RNA, DNA, and Protein Extraction from Biopsies
2.6. DNA Extractions from Whole Blood
2.7. DNA Extractions from Mononuclear Cells
3. Methods
3.1. Blood and Tissue Procurement
3.2. Transport
3.3. RNA, DNA, and Protein Extraction
3.3.1. RNA Extraction from PAXgene Blood Tubes
3.3.2. Globin Reduction of RNA Derived from PAXgene Blood Tubes
3.3.3. Isolation of Mononuclear Cells and Separation of Plasma from CPT Tubes and the Processing of the PPT Tubes
3.3.4. Extraction of RNA, DNA, and Protein from Mononuclear Cells
3.3.5. Extraction of RNA, DNA, and Protein from Kidney Biopsies
3.3.5.1. RNA Extraction: Trizol
3.3.5.2. DNA Extraction: Trizol
3.3.5.3. Protein Extraction: Trizol
3.3.6. DNA Extraction from Whole Blood
3.3.7. DNA Extraction from Mononuclear Cells
4. Notes
References
Chapter 2
Isolation of Total RNA from Transgenic Mouse Melanoma Subsets Using Fluorescence-Activated Cell Sorting
1. Introduction
2. Materials
2.1. Cell Culture and Antibody Staining
2.2. Flow Cytometry and Sorting
2.3. RNA Isolation
3. Methods
3.1. Quality Control of the Fluorescence-Activated Cell Sorter
3.2. Preparation of Melanoma Cells and Antibody Staining for FACS
3.3. Methods for Sorting Cell Subsets for Total RNA Extraction
3.3.1. RNeasy System for RNA Isolation After Centrifugation
3.3.2. Direct Sort Method
3.3.2.1. Direct RNA Extraction Using a Combined Trizol LS-RNeasy Method
3.3.2.2. Direct RNA Extraction Using RNeasy Microcolumn Method
3.4. Analyzing RNA from Sorted Melanoma Cell Fractions
3.4.1. Quantitation of RNA
3.4.2. Assessing RNA Quality
3.4.3. Expected RNA Yields
4. Notes
References
Chapter 3
Microarray Analysis of Embryonic Stem Cells and Differentiated Embryoid Bodies
1. Introduction
2. Materials
2.1. Mouse E14 Feeder-Independent ESC Culture
2.2. Embryoid Body (EB) Formation by Hanging Drops
2.3. Total RNA Extraction
2.4. cDNA Synthesis
2.5. cRNA Synthesis and Labeling
2.6. Genechip Hybridization
2.7. GeneChip Wash, Stain and Scan
3. Methods
3.1. Mouse E14 Feeder-Independent ESC Culture
3.2. Embryoid Body Formation by Hanging Drops
3.3. Total RNA Extraction
3.4. cDNA Synthesis
3.5. cRNA Synthesis and Labeling
3.6. GeneChip Hybridization
3.7. GeneChip Wash, Stain, and Scan
3.8. Quality Control of Hybridized Microarrays
3.9. Generation of Signal Values and Multiple Testing Procedures
4. Notes
References
Chapter 4
Determination of Alternate Splicing Events Using the Affymetrix Exon 1.0 ST Arrays
1. Introduction
2. Materials
2.1. Equipment
2.2. Materials for Cell Culture
2.3. Materials for RNA Isolation
2.4. Materials for RNA QC and Microarray Experiment
3. Methods
3.1. Cell Culture and Harvesting of Cells for RNA Isolation
3.2. RNA Isolation
3.3. Qiagen RNEasy Mini-Cleanup
3.4. Assessment of RNA Quality
3.5. Expression Analysis of mRNA from Cells
3.6. Synthesis of Labeled cDNA and Microarray Hybridization
3.6.1. Ribosomal Reduction of 1 mg Total RNA
3.6.2. Synthesis of Labeled cDNA
3.6.3. Hybridization and Scanning
3.7. Analysis of Human Exon 1.0 ST Array Data
3.7.1. Gene Level Analysis
3.7.2. Exon Splicing Analysis (Core-Level)
4. Notes
References
Chapter 5
Profiling microRNA Expression with the Illumina BeadChip Platform
1. Introduction
2. Materials
2.1. Preparation of Poly-A Polymerase (PAP) Plate
2.2. Preparation of cDNA Synthesis (CSP) Plate
2.3. Preparation of Allele-Specific Extension (ASE) Plate
2.4. Addition of Master Mix for Extension and Ligation (MEL)
2.5. Preparation of PCR Plate
2.6. Inoculation of PCR Plate
2.7. Binding of PCR Products
2.8. Preparation of INT Plate for BeadChip
2.9. Hybridization to BeadChip
2.10. Washing BeadChip
3. Methods
3.1. Preparation of Poly-A Polymerase (PAP) Plate (see Note 5)
3.2. Preparation of cDNA Synthesis (CSP) Plate
3.3. Preparation of Allele-Specific Extension (ASE) Plate
3.4. Addition of Master Mix for Extension and Ligation (MEL)
3.5. Preparation of PCR Plate
3.6. Inoculation of PCR Plate
3.7. Binding of PCR Products
3.8. Preparation of INT Plate for BeadChip
3.9. Hybridize BeadChip
3.10. Wash BeadChip
4. Notes
References
Chapter 6
TaqMan® Array Cards in Pharmaceutical Research
1. Introduction
1.1. TaqMan® Gene Expression Assays
1.2. Experimental Design and Analysis
2. Materials
2.1. Test Materials
2.2. Reagents
2.3. Equipment
3. Methods
3.1. Cell Lysis
3.2. Reverse Transcription
3.3. Real-Time PCR Set Up (see Note 9)
3.4. Real-Time PCR Run
4. Notes
References
Chapter 7
DMET ™ Microarray Technology for Pharmacogenomics-Based Personalized Medicine
1. Introduction
2. Materials
2.1. Laboratory Requirements
2.2. Affymetrix Reagents and Arrays Required
2.2.1. Affymetrix DMET Plus Premier Pack (P/N 901268)
2.2.2. Wash Solutions (Shipped Separately; Store at Room Temperature) Includes
2.2.3. 48 DMET™ Plus Arrays (P/N 901317; Shipped Separately; Store at +4°C)
2.3. Required Reagents from Other Suppliers
2.4. Required Affymetrix Equipment and Software
2.5. DNA Input Requirements
3. Methods
3.1. Introduction
3.2. Preparing Genomic DNA Plate 1 (GP1)
3.3. mPCR
3.4. Annealing and Addition of DMET MIP Panel
3.4.1. Location: Preamplification Lab
3.5. MIP Assay and Amplification
3.5.1. Location: Preamplification Lab
3.6. Clean-up
3.7. Fragmentation
3.8. Labeling
3.9. Hybridization
3.10. Washing, Staining, and Scanning
3.11. Data Analysis
3.12. Use Cases
4. Notes
References
Chapter 8
The Use of Microarray Technology for Cytogenetics
1. Introduction
2. Materials
2.1. General
2.2. DNA Purification
2.3. Digestion of Genomic DNA (If Required)
2.4. DNA Washing and Hybridization
2.5. Washing
3. Methods
3.1. Genomic DNA Purification
3.1.1. DNA Extraction from Whole Blood
3.1.2. Cell Lysis
3.1.3. RNase Treatment
3.1.4. Protein Precipitation
3.1.5. DNA Precipitation
3.1.6. DNA Hydration
3.1.7. DNA Quantification
3.1.8. Sonication of Genomic DNA (Recommended Method)
3.1.9. Digestion of Genomic DNA (Alternative to Sonication)
3.2. Microarray Labeling and Hybridization
3.2.1. DNA Labeling (Using the BioPrime Total Labeling Kit)
3.2.2. Purification of Labeled DNA
3.2.3. Hybridization of the Microarray
3.2.4. Sample Preparation
3.2.5. Microarray Hybridization
3.2.6. Washing the Microarray
3.3. Microarray Scanning and Analysis
3.3.1. Scanning the Microarray
4. Notes
References
Chapter 9
PCR/LDR/Universal Array Platforms for the Diagnosis of Infectious Disease
1. Introduction
2. Materials
2.1. Microarray Printing and Coupling DNA Probes
2.2. Postcoupling and Hybridization
2.3. Gene Specific PCR
2.4. Ligase Detection Reaction (LDR) for Detection of Pathogen
3. Methods
3.1. Printing
and Coupling of Zip-Code Addresses
3.2. Postcoupling Processing
3.3. Array Hybridization Quality Control
3.4. Setup of PCR Amplification
3.5. Setup of LDR
3.6. Hybridization of LDR Products
4. Notes
References
Chapter 10
RIP-CHIP in Drug Development
1. Introduction
2. Materials
2.1. Equipment
2.2. Materials and Reagents for Preparing PLB Lysates from Cells Treated with the Drug of Choice
2.3. Materials and Reagents for RNP-IP (RIP)
2.4. Materials and Reagents for RNA QC and Microarray Profiling
3. Methods
3.1. Treatment of Cells with the Experimental Drug and Preparation of PLB Lysate
3.2. Coating of the Beads with Antibody
3.2.1. Coating Sepharose Beads
3.2.2. Coating Magnetic Beads
3.3. Immuno -Łprecipitation of RNA Binding Protein–mRNA Complex (RIP)
3.4. Purification of RNA
3.5. Assessment of RNA Quality
3.6. Synthesis of Labeled cRNA and Microarray Hybridization
3.7. Analysis of the Microarray Data
4. Notes
References
Chapter 11
ChIPing Away at Global Transcriptional Regulation
1. Introduction
2. Materials
2.1. Cell Culture and Cell Pellet Collection and Lysis
2.2. Chromatin Shearing and Fragment Size Confirmation by Agarose Gel Electrophoresis
2.3. Cell Lysate Preclearing and Incubation with Antibody
2.4. Recovery and Washing of Antibody–Protein–DNA Complexes
2.5. Elution of the Antibody–Protein–DNA Complex
2.6. Decrosslinking the Protein–DNA Complex, RNAse A Digest
2.7. ImmunoprecipiŁtated DNA: Fragment Recovery and Proteinase K Digest
2.8. ImmunoprecipiŁtated DNA: Primer A Annealing and Extension
2.9. ImmunoprecipiŁtated DNA: Primer B Annealing, aa-dUTP Labeling and Amplification
2.10. Amplified Immunoprecipitated DNA: PCR Purification
2.11. Amplified DNA: Alexa Fluor Labeling and Purification
2.12. Microarray Hybridization and Wash Protocol
3. Methods
3.1. Cell Culture and Cell Pellet Collection and Lysis
3.2. Chromatin Shearing and Fragment Size Confirmation by Agarose Gel Electrophoresis
3.3. Cell Lysate Preclearing and Incubation with Antibody
3.4. Recovery and Washing of Antibody–Protein–DNA Complexes (see Note 3)
3.5. Elution of the Antibody–Protein–DNA Complex
3.6. Decrosslinking the Protein–DNA Complex, RNAse A Digest
3.7. ImmunoprecipiŁtated DNA: Fragment Recovery and Proteinase K Digest
3.8. Round A/B Random Amplification of Immunoprecipitated DNA: Primer A Annealing and Extension (see Note 4)
3.9. Round A/B Random Amplification of Immunoprecipitated DNA: Primer B Annealing, aa-dUTP Labeling and Amplification (see
3.10. Amplified Immunoprecipitated DNA: PCR Purification
3.11. Amplified Immunoprecipitated DNA: Alexa Fluor Labeling and Purification
3.12. Microarray Hybridization and Wash Protocol
4. Notes
References
Chapter 12
HELP (HpaII Tiny Fragment Enrichment by Ligation-Mediated PCR) Assay for DNA Methylation Profiling of Primary Normal and Malig
1. Introduction
2. Materials
3. Methods
3.1. DNA Extraction from the Frozen Tissue Samples
3.2. DNA Extraction from the Cell Pellets
3.3. HELP
3.4. Data Analysis
4. Notes
References
Chapter 13
High-Throughput Screening of Metalloproteases Using Small Molecule Microarrays
1. Introduction
2. Materials
2.1. Chemicals and Biochemicals
2.1.1. Combinatorial Synthesis of Small Molecule Library
2.1.2. Fabrication of Avidin Slides
2.1.3. Enzyme Labeling and Screening
2.2. Equipment and Supplies
2.2.1. Combinatorial Synthesis of Small Molecule Library
2.3. Softwares
3. Methods
3.1. Combinatorial Synthesis of Small Molecule Library
3.2. Fabrication of Avidin Slides
3.3. Printing Chemical Libraries on Microarray Slides
3.4. Protein Labelling and Application
4. Notes
References
Chapter 14
Metabolic Enzyme Microarray Coupled with Miniaturized Cell-Culture Array Technology for High-Throughput Toxicity Screening
1. Introduction
2. Materials
2.1. Slide Treatment
2.2. Microarray Spotting
2.3. Cell Culture
2.4. Cell Staining
3. Methods
3.1. Preparation of Functionalized Slides
3.1.1. Acid Cleaning of the Glass Slide
3.1.2. Poly(Styrene-co-Maleic Anhydride) (PS-MA) Coating for Cell Printing
3.1.3. MethyltrimethoxyŁsilane (MTMOS) Coating for Compound and Enzyme Printing
3.1.4. Preparation of a Sol-Gel Gasket on the MTMOS-Coated Slide for Stamping
3.2. Preparation of a Miniaturized Enzyme Array (the Metabolizing Enzyme Toxicology Assay Chip or MetaChip) on the MTMOS-C
3.3. Preparation of Human Cell Suspension for Spotting
3.4. Preparation of a Miniaturized 3D Cell-Culture Array (the Data Analysis Toxicology Assay Chip, or DataChip) on the PS-MA
3.5. Stamping the DataChip onto the MetaChip
3.6. Cell Staining, Scanning, and Data Analysis
4. Notes
References
Chapter 15
Use of Tissue Microarray to Facilitate Oncology Research
1. Introduction
2. Materials
2.1. DNA Extraction from Donor Paraffin Blocks
2.2. Quantitative PCR
2.3. Fluorescent IHC and AQUA Analysis
3. Methods
3.1. Tissue Microarray Construction and DNA Extraction from Specimens
3.2. Quantitative PCR to Determine HPV16 Viral Load
3.3. Fluorescent Immunohisto-chemistry
3.4. Automated Quantitative Protein Expression Analysis (AQUA™)
4. Notes
References
Chapter 16
Small Molecule Selectivity and Specificity Profiling Using Functional Protein Microarrays
1. Introduction
2. Materials
2.1. Materials and Equipment for Tritiated Small Molecule Profiling
2.2. Scanning and Data Analysis
2.3. Protein Microarray Manufacturing
3. Methods
3.1. Tritiated Small Molecule Interaction Profiling on Functional Human Protein Microarrays
3.2. Data Analysis and Hit Identification
4. Notes
References
Chapter 17
Production and Application of Glycan Microarrays
1. Introduction
2. Materials
2.1. Plate Setup
2.2. Printing
2.3. Humidification/Immobilizing
2.4. Numbering, Blocking, and Storage
2.5. Incubation
3. Methods
3.1. Plate Setup
3.2. Printing
3.3. Humidification/Immobilizing
3.4. Numbering, Blocking, and Storage
3.5. Incubation
3.5.1. For All Array Incubations
3.5.2. For Plant Lectins
3.5.3. For Serum
3.5.4. For Viruses
3.6. Scanning and Data Analysis
4. Notes
References