Molecular Toxicology Protocols (Methods in Molecular Biology, 2102)

Preface
Contents
Contributors
Part I: Bioinformatics and Biostatistics
	Chapter 1: Bioenergetic Analyses of In Vitro and In Vivo Samples to Guide Toxicological Endpoints
		1 Introduction
			1.1 In Vitro Bioenergetics
				1.1.1 MitoXpress Xtra Assay
				1.1.2 Live/Dead Assay
				1.1.3 NADH/NADPH
				1.1.4 2-NBDG (2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose)
			1.2 In Vivo Bioenergetics
				1.2.1 Fluorodeoxyglucose (18F)-Positron Emission Tomography (FDG-PET)
				1.2.2 Indocyanine Green (ICG)
		2 Materials
			2.1 Cell Culture and Exposure
				2.1.1 In Vitro Experiments
			2.2 Animal Experiments and Exposure
				2.2.1 In Vivo Experiments
		3 Methods
			3.1 Cell Culture
				3.1.1 MitoXpress Assay
				3.1.2 Live/Dead Assay
				3.1.3 NADH Measurement
				3.1.4 2-NBDG Technique
			3.2 Animal Housing
				3.2.1 18FDG-PET In Vivo Experiment
				3.2.2 ICG-IVIS Spectrum In Vivo
			3.3 Data Analysis
				3.3.1 Selecting Time Points of Interest from Real Time Data Using NADH and Oxygen Consumption to Predict ATP Generation to Det...
				3.3.2 Using Bioenergetics to Guide Toxicological Endpoints
		4 Notes
		References
	Chapter 2: Next Generation Sequencing (NGS) Application in Multiparameter Gene Expression Analysis
		1 Introduction
		2 Materials
			2.1 Growth Media for Human Cell Cultures
			2.2 GRO-Seq Analysis
			2.3 Polysome Profiling Analysis
			2.4 RNA-Seq Analysis
		3 Methods
			3.1 GRO-Seq
				3.1.1 Nuclei Extraction
				3.1.2 NRO Reaction
				3.1.3 DNase Treatment
				3.1.4 Hydrolysis Fragmentation of RNA
				3.1.5 Treatment of NRO-RNA with Antarctic Phosphatase
				3.1.6 Nascent RNA Purification with Anti-BrUTP Beads
				3.1.7 Use the RNA Product for RNA-Seq Analysis
			3.2 Polysome Profiling
				3.2.1 Preparation of the Sucrose Gradient
				3.2.2 Growing Cells and Cycloheximide Treatment
				3.2.3 Cell Lysis, Lysate Clarification, and Gradient Centrifugation
				3.2.4 Ultracentrifugation, Collection of Fractions, and RNA Purification
				3.2.5 Use the RNA Samples for Standard RNA-Seq Analysis
			3.3 RNA-Seq Analysis
				3.3.1 mRNA Purification and Fragmentation
				3.3.2 Synthesis of First Strand cDNA (For GEO-Seq, Start from This Step)
				3.3.3 Second Strand cDNA Synthesis
				3.3.4 cDNA Fragments End Repaired
				3.3.5 3′-End Adenylation
				3.3.6 Adaptor Ligation
				3.3.7 DNA Fragment Enrichment
				3.3.8 DNA Fragment Enrichment
			3.4 Simultaneous GRO-Seq, RNA-Seq, and Polysome Profiling Analyses
		4 Notes
		References
	Chapter 3: Integrated Chip-Seq and RNA-Seq Data Analysis Coupled with Bioinformatics Approaches to Investigate Regulatory Land...
		1 Introduction
		2 Methods
			2.1 Step 1: Acquire or Download ChIP-Seq Experimental Data
			2.2 Step 2: Processing and Analyzing ChIP-seq Data
				2.2.1 Quality Control of Sequencing Reads
				2.2.2 Mapping Sequencing Reads to the Genome (Alignment)
				2.2.3 Peak Calling
				2.2.4 Gene Annotation (Identifying Target Genes)
			2.3 Steps Involved in RNA-Seq Analysis
				2.3.1 Quality Control of RNA Sequence Reads
				2.3.2 Mapping (Generation of Alignments)
				2.3.3 Transcript Quantification
				2.3.4 Identification of Differential Expressed (DE) Genes
			2.4 Integration of ChIP-Seq and RNA-Seq Data
		3 Exercise of Integrating NRF1 ChIP-Seq and RNA-Seq Data
			3.1 Activating and Repressive Function Prediction
			3.2 Direct NRF1 Target Prediction
			3.3 Binding Motif Analysis
			3.4 Interpretation of Integrated Analysis
		4 Conclusion
		References
	Chapter 4: Analysis of RNA Sequencing Data Using CLC Genomics Workbench
		1 Introduction
		2 Materials
			2.1 RNA-Seq Dataset
			2.2 Download CLC Genomics Workbench 12 (See Note 1)
			2.3 System Requirement
			2.4 Workbench Licenses
			2.5 Plugin Installation
			2.6 CLCBio Genomics Workbench Server
			2.7 Ingenuity Pathway Analysis (IPA)
		3 Methods
			3.1 CLC Server Connection (Optional)
			3.2 RNA Sequencing Data Analysis Workflow
			3.3 Import FASTQ Reads to CLC Genomics Workbench
			3.4 Quality Control of Sequencing Reads
			3.5 Trim Reads (See Note 4)
			3.6 Import Metadata Table
			3.7 Reads Mapping
			3.8 Create a PCA Plot
			3.9 Differential Expression
			3.10 Create a Heatmap
			3.11 Create a Venn Diagram
			3.12 Downstream Analysis
		4 Notes
		References
Part II: Toxicant-Induced Lung Inflammation and Lung Tumors
	Chapter 5: Using Bronchoalveolar Lavage to Evaluate Changes in Pulmonary Diseases
		1 Introduction
		2 Materials
			2.1 Reagents for Lavage, Anesthesia, and Fixation
			2.2 Dissection Tools, Materials, and Instruments
		3 Methods
			3.1 Bronchoalveolar Lavage (BAL)
			3.2 Inflated Fixation of Lung Tissues
			3.3 Cell Differential Counts
		4 Notes
		References
	Chapter 6: Fast and Efficient Measurement of Clinical and Biological Samples Using Immunoassay-Based Multiplexing Systems
		1 Introduction
		2 Materials
			2.1 Samples and Reagents
				2.1.1 Plasma and Serum Collection
				2.1.2 Tissue Lysate
			2.2 Instrument
		3 Methods
			3.1 Sample Collection
				3.1.1 Serum Collection
				3.1.2 Plasma Collection
				3.1.3 Human BAL Fluid
				3.1.4 Mouse BAL Fluid
				3.1.5 Human Lung Tissue Lysate
				3.1.6 Mouse Lung Tissue Lysate
			3.2 Luminex Multiplex Analysis
				3.2.1 Preparation for the Luminex Assay
				3.2.2 Preparation of Standards
				3.2.3 Preparation of Assay Samples
				3.2.4 Preparation of Antibody-Coated Beads (Magnetic and Polystyrene)
				3.2.5 Incubation of Test Samples with Antibody-Coated Polystyrene Beads
				3.2.6 Alternative Protocol for Using Magnetic Beads
				3.2.7 Incubation of Detection Antibody
				3.2.8 Incubation of Streptavidin-PE Solution
				3.2.9 Detection of Florescent Intensity of the Assayed Samples with the Luminex Machine
				3.2.10 Data Acquisition
				3.2.11 Data Analysis
				3.2.12 Bioplex Maintenance
			3.3 MSD Multiplex Analysis
				3.3.1 Preparation for the MSD Assay
				3.3.2 Preparation of Standards
				3.3.3 Preparation of Assay Samples
				3.3.4 Preparation of Antibody-Coated Plate
				3.3.5 Incubation of Test Samples and Standards
				3.3.6 Incubation of Detection Antibody
				3.3.7 Reading of the Assay Plate
				3.3.8 Data Acquisition
				3.3.9 Data Analysis
		4 Notes
		References
	Chapter 7: Induction of Lung Tumors and Mutational Analysis in FVB/N Mice Treated with the Tobacco Carcinogen 4-(Methylnitrosa...
		1 Introduction
		2 Materials
			2.1 Animals
			2.2 Chemicals and Reagents
			2.3 Animal Procedure Equipment
			2.4 Fixative Apparatus Equipment
			2.5 Tumor Count Equipment
			2.6 Paraffin Embedding and Immunohistochemistry Equipment
			2.7 Isolation of Tumor DNA and Mutation Analysis
		3 Methods
			3.1 NNK Administration
			3.2 Lung Catheterization
			3.3 Lung Inflation and Fixation
			3.4 Lung Tumor Assessment
				3.4.1 Tumor Count
				3.4.2 Lung Tumor Size Measurement
			3.5 Mutation Analysis
				3.5.1 Paraffin Block Preparation and H&E Staining
				3.5.2 Isolation of Tumor DNA and Mutation Analysis
		4 Notes
		References
Part III: Analysis of Protein and Gene Expression and Silencing
	Chapter 8: Two-Dimensional Difference Gel Electrophoresis: A Gel-Based Proteomic Approach for Protein Analysis
		1 Introduction
		2 Materials
			2.1 Sample Preparation
			2.2 Protein Quantitation
			2.3 Labeling
			2.4 Rehydration
			2.5 First Dimension
			2.6 Equilibration
			2.7 Second Dimension
			2.8 Post-Gel Analyses
		3 Methods
			3.1 Sample Preparation
				3.1.1 Cultured Cells
				3.1.2 Tissues
				3.1.3 Body Fluids
			3.2 Protein Quantitation
			3.3 Labeling
				3.3.1 Reconstitution of CyDye in DMF
				3.3.2 Preparation of CyDye Working Solution
				3.3.3 Minimal Labeling a Protein Sample
			3.4 Rehydration (See Note 13)
			3.5 First Dimension-Isoelectric Focusing
			3.6 Equilibration
			3.7 Second Dimension-SDS-PAGE
				3.7.1 Preparation of SDS-PAGE (See Note 22)
				3.7.2 SDS-PAGE
			3.8 Post-Gel Analyses
		4 Notes
		References
	Chapter 9: Determination and Quantification of Bacterial Virulent Gene Expression Using Quantitative Real-Time PCR
		1 Introduction
		2 Materials
		3 Methods
			3.1 RNA Sample Preparation
			3.2 Reverse Transcription (RT)
			3.3 Real-Time Gene Quantification (qRT-PCR)
			3.4 Data Analysis
		4 Notes
		References
	Chapter 10: Using a Lentivirus-Based Inducible RNAi Vector to Silence a Gene
		1 Introduction
		2 Materials
			2.1 Equipment
			2.2 Reagents
			2.3 Cell Culture
			2.4 Supplies
		3 Methods
			3.1 sh-TOP1 Oligo Design
			3.2 Inducible pLV-H1TetO-sh-TOP1-GFP-Bsd Vector Cloning
			3.3 Inducible pLV-H1TetO-sh-TOP1-GFP-Bsd Plasmid Preparation and Oligo Insertion Confirmation
			3.4 shRNA Lentivirus Production Using the Recombinant pLV-H1TetO-sh-TOP1-GFP-Bsd Vectors
			3.5 shRNA Stable Cell Line Establishment
			3.6 Knockdown Efficiency Analysis of the Inducible Stable Cell Lines
		4 Notes
		References
Part IV: Gene Promoter Methylation
	Chapter 11: Analysis of Toxicants-Induced Alterations in DNA Methylation by Methylation-Sensitive-Random Amplified Polymorphic...
		1 Introduction
		2 Materials
			2.1 DNA Isolation from Mammalian Cells or Tissue
			2.2 Restriction Enzyme Digestion of DNA
			2.3 MS-RAPD-PCR Amplification
			2.4 Agarose Gel Electrophoresis
		3 Methods
			3.1 DNA Isolation from Mammalian Cells or Tissue
			3.2 Digestion of Genomic DNA with Methylation-Sensitive Restriction Enzymes (MspI and HpaII)
			3.3 MS-RAPD-PCR Amplification
			3.4 Agarose Gel Electrophoresis of MS-RAPD-PCR Amplification Products
		4 Notes
		References
	Chapter 12: Detection of DNA Methylation by MeDIP and MBDCap Assays: An Overview of Techniques
		1 Introduction
		2 Materials
			2.1 Instruments
			2.2 Reagents
				2.2.1 MeDIP
				2.2.2 MBDCap
		3 Methods
			3.1 MeDIP
				3.1.1 Preparation of Genomic DNA
				3.1.2 Sonication of Genomic DNA
				3.1.3 Immunoprecipitation of Methylated DNA (MeDIP)
				3.1.4 Purification of Methylated DNA
			3.2 MBDCap Assay by MethylMiner Kit
				3.2.1 Initial Bead Wash
				3.2.2 Coupling the MBD-Biotin Protein to the Beads
				3.2.3 Wash the MBD-Beads
				3.2.4 Fragmented DNA by Sonicating Genomic DNA
				3.2.5 Capture Reaction
				3.2.6 Removing Non-captured DNA from the Beads
				3.2.7 Single Fraction Elution
				3.2.8 Ethanol Precipitation (DNA Cleanup)
			3.3 Experimental Analyses
				3.3.1 Locus-Specific Validation
				3.3.2 Genome-Wide Profiling
		4 Notes
		References
Part V: Detection of Chromosomal and Genome-Wide Damage
	Chapter 13: Analysis of Telomere Length and Aberrations by Quantitative FISH
		1 Introduction
		2 Materials
			2.1 Preparation of Metaphases
			2.2 Metaphase Spreads
			2.3 Cell Preparation for FISH
			2.4 FISH
			2.5 Washes and Mounting
			2.6 Acquisition and Analysis
		3 Methods
			3.1 Preparing Metaphases
			3.2 Metaphase Spreads
			3.3 Cell Preparation for FISH
			3.4 FISH
			3.5 Washes and Mounting
			3.6 Acquisition
			3.7 Analysis of Telomeric Aberrations
			3.8 Analysis of Telomere Shortening
		4 Notes
		References
	Chapter 14: Detection of Loss of Heterozygosity in Tk-Deficient Mutants from L5178Y Tk+/--3.7.2C Mouse Lymphoma Cells
		1 Introduction
		2 Materials
			2.1 Equipment
			2.2 Chemical Agents
			2.3 Supplies
		3 Methods
			3.1 Collection of Tk Mutants
			3.2 Cell Lysis and DNA Extraction
			3.3 Microsatellite Marker Identification
			3.4 Allele-Specific PCR
			3.5 Gel Electrophoresis
			3.6 Data Analysis
		4 Notes
		References
	Chapter 15: Detection of DNA Double-Strand Breaks and Chromosome Translocations Using Ligation-Mediated PCR and Inverse PCR
		1 Introduction
			1.1 Ligation-Mediated PCR
			1.2 Inverse PCR
			1.3 Parallel Sequencing
			1.4 A Representative Translocation in MLL
		2 Materials
			2.1 Ligation-Mediated PCR
				2.1.1 Construction of the Asymmetric Double-Strand Linker
				2.1.2 Purification and Preparation of Template DNA
				2.1.3 Seminested PCR Amplification of Linker-Ligated DNA
				2.1.4 Southern Blot Analysis of PC-Amplified DNA
			2.2 Inverse PCR
				2.2.1 Preparation and Purification of the DNA Template
				2.2.2 Restriction Enzyme Digestion
				2.2.3 Ligation Reaction
				2.2.4 Inverse PCR
				2.2.5 Cloning and Sequencing of IPCR Products
				2.2.6 Translocation Sequence Analysis
			2.3 Parallel Sequencing
				2.3.1 IPCR
				2.3.2 Sonication
				2.3.3 End Repair
				2.3.4 Adding 3′ Adenosines
				2.3.5 Ligation of Adapters
				2.3.6 Purification of Ligation Products
				2.3.7 Enrich the Adapter-Modified DNA Fragments by PCR
		3 Methods
			3.1 Ligation-Mediated PCR
				3.1.1 Construction of the Asymmetric Double-Strand Linker
				3.1.2 Purification and Preparation of Template DNA
				3.1.3 Semi-Nested PCR Amplification of Linker-Ligated DNA
			3.2 Inverse PCR
				3.2.1 Preparation and Purification of the DNA Template
				3.2.2 Restriction Enzyme Digestion
				3.2.3 Ligation Reaction
				3.2.4 Inverse PCR
				3.2.5 Cloning of Inverse PCR Products
				3.2.6 Translocation Sequence Analysis
			3.3 Parallel Sequencing
				3.3.1 Sample Sonication
				3.3.2 Sample DNA End Repair (See Note 9)
				3.3.3 Alternative DNA End Repair Using NEB End Repair Module
				3.3.4 Addition of Adenine to the 3′ End of DNA Fragments
				3.3.5 Ligate Adapters to DNA Fragments
				3.3.6 Ligation Product Purification
				3.3.7 Amplification of the Adapter-Modified DNA Fragments
		4 Notes
		References
Part VI: Analysis of DNA-Adducts and Surrogate Gene Mutations
	Chapter 16: 32P-Postlabeling Analysis of DNA Adducts
		1 Introduction
		2 Materials
			2.1 DNA Digestion
			2.2 Nuclease P1 Digestion
			2.3 Butanol Extraction
			2.4 DNA Postlabeling
			2.5 Thin-Layer Chromatography
			2.6 Detection and Quantification
			2.7 HPLC Cochromatography
		3 Methods
			3.1 DNA Digestion
			3.2 Nuclease P1 Digestion
			3.3 Butanol Extraction
			3.4 Labeling of Adducts (See Note 8)
			3.5 Test for Efficiency of Enrichment Techniques
			3.6 Determining the Specific Activity of [γ-32P]ATP
			3.7 Imaging and Quantification
			3.8 Extraction of Adducts for HPLC Cochromatography
			3.9 HPLC Cochromatography
		4 Notes
		References
	Chapter 17: Determination of Mutational Spectra Induced by Environmental Toxicants in Complex Human Cell Populations
		1 Introduction
		2 Materials
			2.1 Cell Lines, Culture Media, and Apparatus
			2.2 Chemicals
			2.3 Reagents for Molecular Analysis
			2.4 Primer Labeling
			2.5 Polyacrylamide Gel Electrophoresis (PAGE)
			2.6 Denaturing Gradient Gel Electrophoresis (DGGE)
		3 Methods
			3.1 Cell Culture, Chemical Treatment, and 6TG Selection
				3.1.1 Cell Culture
				3.1.2 Treatment of Cells
				3.1.3 6TG Selection of HPRT Mutants
			3.2 Analysis of Mutations in HPRT Mutants
				3.2.1 PCR Conditions
				3.2.2 DGGE Analysis
				3.2.3 Determination of the Kind and Position of Mutations
		4 Notes
		References
	Chapter 18: Detection of Pig-a Mutant Erythrocytes in the Peripheral Blood of Rats and Mice
		1 Introduction
		2 Materials
			2.1 Blood Collection
			2.2 Blood Cell Labeling
			2.3 Blood Processing and Analysis
		3 Methods
			3.1 Blood Collection
				3.1.1 From Rat Tail Vein (See Note 3)
				3.1.2 From Mouse Saphenous Vein
			3.2 Blood Cell Labeling
				3.2.1 Labeling of Rat Blood (See Notes 1 and 4)
				3.2.2 Labeling of Mouse Blood
			3.3 Flow Cytometer Setup Using a FACSAria I Flow Cytometer and FACSDiva 6.1.1 Software
			3.4 Sample Analysis
		4 Notes
		References
	Chapter 19: Analysis of In Vivo Mutation in the Hprt and Tk Genes of Mouse Lymphocytes
		1 Introduction
		2 Materials
			2.1 Medium for Culture and Propagation of T Lymphocytes (See Note 2 and Ref. 37)
			2.2 Isolation and Priming of T-Lymphocytes
			2.3 Lymphocyte Primary Culture
			2.4 Scoring Clones and Determining Mutant Frequencies
			2.5 Molecular Analysis of Mutations
		3 Methods
			3.1 Isolation of Lymphocytes
			3.2 Limiting Dilution Culture of Lymphocytes
			3.3 Scoring Lymphocyte Clones in 96-Well Plates
				3.3.1 Manual Method
				3.3.2 Automated Method
			3.4 Calculating Mutant Frequencies
			3.5 Preservation of Cells for Molecular Analysis
			3.6 Expansion of 6TG-Resistant Lymphocytes Beyond 96-Well Plates
			3.7 Molecular Analysis of Isolated Mutants
				3.7.1 Tk LOH Analysis
				3.7.2 Hprt and Tk RT-PCR Analysis (See Note 11)
				3.7.3 RT-PCR Analysis of Expanded Hprt Mutant Clones
		4 Notes
		References
	Chapter 20: Molecular Analysis of Mutations in the Human HPRT Gene
		1 Introduction
		2 Materials
			2.1 cDNA Synthesis
			2.2 PCR Amplification of the HPRT cDNA
			2.3 HPRT Gene Multi-Exon Analysis
		3 Methods
			3.1 cDNA Synthesis
			3.2 Nested PCR Amplification of the HPRT cDNA
			3.3 HPRT Gene Multi-Exon Analysis
		4 Notes
		References
	Chapter 21: Simultaneous Quantification of t(14;18) and HPRT Exon 2/3 Deletions in Human Lymphocytes
		1 Introduction
		2 Materials
			2.1 Lymphocyte Isolation
			2.2 DNA Isolation (See Note 1)
			2.3 PCR Assay (See Note 2)
		3 Methods
			3.1 Lymphocyte Isolation (See Note 5)
			3.2 DNA Isolation
			3.3 PCR Assay (See Note 7)
			3.4 Confirmation of t(14;18) or HPRT Exon 2/3 Deletion
		4 Notes
		References
Part VII: Detection and Characterization of Cancer Gene Mutation
	Chapter 22: Analysis of Mutations in K-ras and p53 Genes in Sputum and Plasma Samples
		1 Introduction
		2 Materials
			2.1 Epithelial Cell Isolation from Sputum Samples
			2.2 DNA Extraction from Laser-Captured Cells (See Note 1)
			2.3 Preparation of Plasma and Isolation of Cell-Free DNA
			2.4 DNA Amplification by PCR
			2.5 Restriction Enzyme Digestion
			2.6 Polyacrylamide Gel Electrophoresis (PAGE)
			2.7 Denaturing Gradient Gel Electrophoresis (DGGE)
			2.8 Single-Stranded Conformational Polymorphism (SSCP)
		3 Methods
			3.1 Preparation of Plasma
			3.2 Isolation of Cell-Free DNA from Plasma
			3.3 Isolation of Epithelial Cells by Laser-Capture Microdissection and Mutation Analysis
				3.3.1 Epithelial Cell Isolation from Sputum Samples
				3.3.2 DNA Extraction from Laser-Captured Cells
				3.3.3 Analysis of K-ras Exon 1 Mutations in Laser-Captured Cells by PCR and DGGE
				3.3.4 Analysis of p53 Mutations in Laser-Captured Cells by PCR and SSCP
			3.4 Analysis of Mutations in DNA Extracted from Whole Sputum Cells and in Cell-Free DNA from Plasma
				3.4.1 DNA Extraction from Whole Sputum Samples (See Note 7)
				3.4.2 Analysis of K-ras Mutations by PCR+MAE+DGGE
				3.4.3 Analysis of p53 Mutations by PCR+MAE+PAGE
		4 Notes
		References
	Chapter 23: ACB-PCR Quantification of Low-Frequency Hotspot Cancer-Driver Mutations
		1 Introduction
		2 Materials
			2.1 Genomic DNA Isolation
			2.2 Preparation of PIK3CA Codon 1047 Wild-Type and Mutant Reference DNAs (See Note 5)
			2.3 Amplification of PIK3CA Codon 1047 First-Round PCR Products from Genomic DNA Samples (See Note 5)
			2.4 Purification of PIK3CA Codon 1047 Wild-type and Mutant Reference DNAs and First-Round PCR Products (by Either of Two Metho...
				2.4.1 Ion-Pair Reverse-Phase High-Performance Liquid Chromatography Purification (WAVE) of Wild-Type and Mutant Reference DNAs...
				2.4.2 Agarose Gel Purification of Wild-Type and Mutant Reference DNAs and First-Round PCR Products
			2.5 Quantification of PIK3CA Codon 1047 Wild-Type and Mutant Reference DNAs and First-Round PCR Products
			2.6 Preparation of Mutant Fraction Standards and First-Round PCR Products for ACB-PCR (See Note 7)
			2.7 Preparation of ACB-PCR Reaction Mix and ACB-PCR Reactions
			2.8 Sample and Data Analysis
		3 Methods
			3.1 Genomic DNA Isolation
				3.1.1 Tissue Homogenization and DNA Extraction
				3.1.2 RNAse Digestion and DNA Extraction
				3.1.3 Restriction Enzyme Digestion and DNA Extraction
			3.2 Preparation of Wild-Type and Mutant Reference DNAs
				3.2.1 Linearization of Wild-Type and Mutant Reference DNAs
				3.2.2 Amplification of Wild-Type and Mutant Reference DNAs
			3.3 Amplification of First-Round PCR Products from Genomic DNA Samples
			3.4 Purification of PIK3CA Codon 1047 Wild-Type and Mutant Reference DNAs and First-Round PCR Products
				3.4.1 WAVE Purification of PIK3CA Codon 1047 Wild-Type and Mutant Reference DNAs and First-Round PCR Products
				3.4.2 Agarose Gel Purification of PIK3CA Codon 1047 Wild-Type and Mutant Reference DNAs and First-Round PCR Products
			3.5 Quantification of PIK3CA Codon 1047 Wild-Type and Mutant Reference DNAs and First-Round PCR Products
			3.6 Preparation of MF Standards and First-Round PCR Products for ACB-PCR (See Note 18)
			3.7 Preparation of ACB-PCR Reaction Mix and ACB-PCR Reactions (See Note 21)
			3.8 Sample and Data Analysis
			3.9 ACB-PCR Conditions for Other Mutational Targets
		4 Notes
		References
	Chapter 24: Detection and Characterization of Oncogene Mutations in Preneoplastic and Early Neoplastic Lesions
		1 Introduction
			1.1 Detection of K-RAS Oncogene Mutations in Normal-Appearing (Nonneoplastic) Tissues of Colorectal Cancer Patients
			1.2 Detection of Mutant K-RAS Oncogenes in Aberrant Crypt Foci of Human Colon
		2 Materials
			2.1 Control DNA for Validation of EPCR
			2.2 Preparation of Tissue Samples and DNA Extraction
			2.3 Enriched PCR
			2.4 Polyacrylamide Gel Electrophoresis (PAGE)
		3 Methods
			3.1 Preparation of Mutant DNA Used for Validation of the Sensitivity of EPCR
			3.2 DNA Extraction from Fresh Tissue Samples
			3.3 Extraction of DNA from Formalin-Fixed ACF
			3.4 Enriched PCR
			3.5 Detection of Mutant K-RAS by RFLP Analysis
			3.6 Characterization of Mutant K-RAS Bands Detected by EPCR
		4 Notes
		References
Part VIII: Analysis of DNA Damage and Repair Mechanisms
	Chapter 25: The Sister Chromatid Exchange (SCE) Assay
		1 Introduction
		2 Materials
			2.1 Growing Cells
			2.2 Cellular BrdU Incorporation
			2.3 Harvesting Cells for Metaphase Spreads
			2.4 Preparing and Storing Slides for Metaphase Spreads
			2.5 Differential SCE with Giemsa
		3 Methods
			3.1 Thawing Cryopreserved Cells
			3.2 Expanding the Cell Population and Establishing Doubling Time
			3.3 BrdU Incorporation
			3.4 Arresting Cells in Metaphase
			3.5 Harvesting Cells for Metaphase Spreads
			3.6 Preparing and Storing Slides for Metaphase Spreads
			3.7 Preparing Metaphase Spreads
			3.8 Differential Sister Chromatid Staining with Giemsa
			3.9 Preparing Slides for Visualization
			3.10 Visualizing and Digital Imaging of Chromosomes
			3.11 Scoring SCEs and Analyzing Data
		4 Notes
		References
	Chapter 26: The Gene Cluster Instability (GCI) Assay for Recombination
		1 Introduction
			1.1 Expected Results
		2 Materials
			2.1 Thawing Cryopreserved Cells
			2.2 Subculturing Cells for GCI Analysis
			2.3 Deriving and Expanding Clonal Lines
			2.4 Preparing Subcultured Cells for DNA Extraction
			2.5 Isolation of Solid-Phase High-Molecular-Weight Genomic DNA from Human Cells
			2.6 Enzymatic Digestion of High-Molecular-Weight DNA for PFGE
			2.7 Preparation of Agarose Gel for PFGE
			2.8 Loading Digested DNA Samples into the Agarose Gel for PFGE
			2.9 Loading and Running Pulsed-Field Electrophoresis Gels
			2.10 Ethidium Staining and Preparing the Gel for In-Gel Hybridization of a Radiolabeled Probe
			2.11 PCR Reaction: Non-Radiolabeled Southern Blot Probe Template Preparation and Radiolabeled 45S rDNA Southern Blot Probe Gen...
			2.12 Southern Blot Analysis Using In-Gel Hybridization of the Radiolabeled rDNA Probe
		3 Methods
			3.1 Thawing Cryopreserved Cells
			3.2 Subculturing Cells
			3.3 Deriving and Expanding Clonal Lines
			3.4 Preparing Cultured Cells for High-Molecular-Weight Solid-Phase DNA Isolation
			3.5 Enzymatic Digestion of High-Molecular-Weight DNA for PFGE
			3.6 Preparation of an Agarose Gel for PFGE
			3.7 Loading Digested DNA Samples into the Agarose Gel for PFGE
			3.8 Loading and Running the Bio-Rad CHEF MAPPER Apparatus for PFGE
			3.9 Ethidium Staining and Drying the Gel in Preparation for In-Gel Hybridization with a Radio-Labeled Probe
			3.10 Preparing the Template DNA for PCR Radiolabeling
			3.11 Preparing the Radiolabeled 45S rDNA Probe
			3.12 In-Gel Hybridization of the Radiolabeled rDNA Probe and Southern Analysis
		4 Notes
		References
	Chapter 27: New Perspectives on Unscheduled DNA Synthesis: Functional Assay for Global Genomic DNA Nucleotide Excision Repair
		1 Introduction
		2 Materials
			2.1 Cell Irradiation, Labeling, and Fixation
			2.2 Slide Processing I: Dipping in Emulsion
			2.3 Slide Processing II: Developing Emulsion
			2.4 Grain Counting and Normalization
		3 Methods
			3.1 Cell Irradiation, Labeling, and Fixation
			3.2 Slide Processing I: Dipping in Emulsion
			3.3 Slide Processing II: Developing Emulsion
			3.4 Grain Counting and Normalization
		4 Notes
		References
	Chapter 28: Host Cell Reactivation: Assay for Actively Transcribed DNA (Nucleotide Excision) Repair Using Luciferase Family Ex...
		1 Introduction
		2 Materials
			2.1 Preparation of Host Cells
			2.2 UV Irradiation of Reporter Plasmid
			2.3 Transient Transfection (Lipofection)
			2.4 Protein Isolation
			2.5 Protein Quantification
			2.6 Quantification of Luciferase Expression
			2.7 Quantification of ß-Galactosidase Expression
		3 Methods
			3.1 Preparation of Host Cells
			3.2 UV Irradiation of Reporter Plasmid (See Note 7)
			3.3 Transient Transfection (Lipofection)
			3.4 Protein Harvest and Quantification
			3.5 Protein Quantification
			3.6 Quantification of Luciferase Expression
			3.7 Quantification of ß-Galactosidase Expression (See Note 11)
			3.8 Calculating Relative TCR Capacities
		4 Notes
		References
Part IX: Analysis of Endoplasmic Reticulum Stress and miRNA/RNA Interaction
	Chapter 29: Methods for Establishing and Using a Stable Cell Line Expressing Both Gaussia Luciferase and Firefly Luciferase to...
		1 Introduction
		2 Materials
			2.1 Equipment
			2.2 Cell Culture
			2.3 Reagents for Lentivirus Production, Titration, and Transduction
			2.4 Reagents for Validation and Testing
			2.5 Supplies
		3 Methods
			3.1 Lentivirus Production
				3.1.1 Material Preparation and Cell Culture
				3.1.2 Lentivirus Production in 293T Cells
				3.1.3 Optional Procedure: Virus Concentration and Purification
			3.2 Titering the Lentiviral Stocks
				3.2.1 Reagent Preparation and Cell Culture
				3.2.2 Lentivirus Titration
			3.3 Lentiviral Transduction for Gene Expression and Establishment of Stable Cell Line
				3.3.1 Establishing the Fluc-Expressing Stable HepG2 Cell Line HepG2-Fluc
				3.3.2 Transduction of Gluc-Expressing Lentivirus and Establishing HepG2-Flu2-Gluc Stable Cell Line
				3.3.3 Storing the Cells
			3.4 Validation and Application
				3.4.1 Validation of the Correlation Between Cell Density and Fluc Activity
				3.4.2 Validate the Stable HepG2-Fluc-Gluc Cell Line for Detecting ER Stress Inducers
				3.4.3 Use the Stable Cell Line to Detect Compounds and Drugs for Liability in ER Stress
		4 Notes
		References
	Chapter 30: FREMSA: A Method That Provides Direct Evidence of the Interaction between microRNA and mRNA
		1 Introduction
		2 Materials
			2.1 Biological Samples
			2.2 RNA Oligonucleotides
			2.3 Apparatuses
			2.4 Chemical Solutions
		3 Methods
			3.1 Sample Preparation
				3.1.1 In Silico Analyses
				3.1.2 Oligonucleotides Synthetization
				3.1.3 Oligonucleotides Preparation
				3.1.4 Cytoplasmic Extracts
				3.1.5 Native Polyacrylamide Gel
			3.2 Incubation and Interaction
			3.3 Separation of Probes by Gel Electrophoresis
			3.4 Visualization and Detection
			3.5 Explanation of Results
			3.6 Advantages and Further Consideration
		4 Notes
		References
Correction to: Next Generation Sequencing (NGS) Application in Multiparameter Gene Expression Analysis
Index