Protocols in Advanced Genomics and Allied Techniques ()

Preface
Contents
About the Author
Chapter 1: Conventional and Basic Genomic Techniques
	1 Genomic DNA Isolation
	Points to Remember
		1.1 Genomic DNA Isolation from Mammalian Blood
			1.1.1 Basic Principle
			1.1.2 Protocol
			1.1.3 Flow Chart
		1.2 Salt-Extraction Procedure for Extraction of High-Quality Genomic DNA for PCR-Based Techniques
			1.2.1 Protocol
			1.2.2 Bacterial Genomic DNA Isolation
				Miniprep of Bacterial Genomic DNA
			1.2.3 Materials
			1.2.4 Procedure
				Miniprep of Bacterial Genomic DNA
				Large-Scale CsCl Prep of Bacterial Genomic DNA
				Large-Scale CsCl Prep of Bacterial Genomic DNA (In Short)
				Reagents and Solutions
		1.3 DNA Isolation with Magnetic Beads
	2 Plasmid DNA Isolation from Escherichia coli Culture
	Points to Remember
		2.1 Basic Principle of the Process
		2.2 Protocol
		2.3 Application of Plasmid Isolation
	3 Horizontal Agarose Gel Electrophoresis
		3.1 Principle
		3.2 Protocol
		3.3 Application
	4 Vertical Polyacrylamide Gel Electrophoresis
		4.1 Principle
		4.2 Silver Staining in PAGE
			4.2.1 Detection of DNA in PAGE
				Silver Staining of the Polyacrylamide Gel
		4.3 Application
	5 Quality Checking of DNA
	Points to Remember
		5.1 Checking of Quality, Purity and Concentration of DNA
		5.2 Evaluation of DNA Extraction
			5.2.1 Spectrophotometer and Qubit Measurements
		5.3 Spectrophotometry
			5.3.1 Purity of Genomic DNA
			5.3.2 Concentration of Genomic DNA
			5.3.3 Purity of Genomic DNA
			5.3.4 Concentration of Genomic DNA
		5.4 Qubit Fluorometer
			5.4.1 Gel Electrophoresis
				Quality of Genomic DNA
			5.4.2 Real-Time PCR
	6 Restriction Fragment Length Polymorphism (RFLP)
	Points to Remember
		6.1 Basic Principle
			6.1.1 Digestion Reaction Mixture
			6.1.2 List of Restriction Enzymes Along with Their Cutting Site
			6.1.3 Types of Restriction Enzyme
	7 Single-Stranded Conformation Polymorphism
	Points to Remember
		7.1 Basic Principle
		7.2 Protocol
	References
Chapter 2: Recombinant DNA Technology
	1 Polymerase Chain Reaction
	Points to Remember
		1.1 Principle
		1.2 Protocol with Suitable Example
			1.2.1 Gene Amplification by Polymerase Chain Reaction: A Case Study
				PCR Reaction Mixture
				PCR Program
			1.2.2 Types of PCR
			1.2.3 Application
		1.3 Primer Designing
	2 Extraction of Total RNA
	Points to Remember
		2.1 Basic Principle
		2.2 Protocol
		2.3 Other Methods for RNA Extraction
		2.4 Protocol
			2.4.1 Second DNase I Treatment
			2.4.2 Application
	3 Checking of Quality, Concentration, and Integrity of RNA
	Points to Remember
		3.1 Basic Principle
		3.2 Protocol
			3.2.1 Using Spectrophotometry
			3.2.2 Using Electrophoresis
	4 cDNA Synthesis
	Points to Remember
		4.1 Basic Principle
		4.2 Protocol
			4.2.1 Application
	5 Purification of cDNA
	Points to Remember
		5.1 Basic Principle
		5.2 Protocol
	6 Recombinant DNA Technology and Gene Cloning
	Points to Remember
		6.1 Recombinant DNA Technology
			6.1.1 Basic Principle
		6.2 Protocol for Gene Cloning
	References
Chapter 3: Mitochondrial Genetics
	Points to Remember
	1 Basic Principle of Mitogenetics
	2 The Basic Differences Between Mitochondrial and Nuclear DNA
	3 Some Interesting Facts About Mitochondria
	4 Protocol for Mitochondrial DNA Isolation
		4.1 Protocol for Isolation of Mitochondria
			4.1.1 Models for Investigation
			4.1.2 Isolation of Mitochondria
			4.1.3 Mitochondria Isolation
			4.1.4 Cytochrome c Assay Using an RandD Rat/Mouse Cytochrome c Quantikine ELISA Kit
			4.1.5 Assessment of Mitochondrial (Dys)function
		4.2 Application of Mitochondrial DNA
		4.3 Mitochondria and Diseases
		4.4 Mitochondria and Evolution
	5 Studies on Cytochrome B in Pig and Sheep in Relation to Reproduction and Diseases: A Case Study
		5.1 Animals
		5.2 Cytochrome B Genotyping
		5.3 SNP Detection of Cytochrome B Gene in Garole Sheep
			5.3.1 Three-Dimensional Structure Prediction and Model Quality Assessment
			5.3.2 Protein-Protein Interaction Network Depiction
			5.3.3 Prediction of Post-translational Modification Sites in Mutant Cytochrome B
			5.3.4 Health Examination of the Animals
				Phylogenetic Relationship
				Statistical Analysis
		5.4 Mitochondrial Replacement Therapy
		5.5 Three Parent Baby Concept
			5.5.1 Pronuclear Transfer of Mitochondrial Replacement Therapy
				Limitations of the Technique
			5.5.2 Spindle Transfer
				Limitations of the Technique
	6 The Relevance of Three Parent Offsprings in Animal Science
		6.1 Marker-Assisted Selection
		6.2 Mitochondrial Replacement Therapy or Three Parent Baby Concept
	7 Whole Mitochondrial Genome Sequencing
	8 Numtogenesis
		8.1 Role of Numtogenesis for the Development of Cancer: A Case Study
		8.2 Mitochondrial DNA Escape Through BAK/BAX Macropores: Implications in Numtogenesis Underlying Aging and Cancer-A Case Study
	9 Most Recent and Interesting Finding
	References
Chapter 4: Cloning
	Points to Remember
	1 Basic Principle of Cloning
	2 Cloning Procedures
		2.1 Embryo Cloning
		2.2 Adult Cloning (Reproductive Cloning)
		2.3 Adult Cell Cloning
			2.3.1 Merits and Demerits of Adult Cell Cloning
				Merits
				Demerits
	3 Somatic Cell Cloning Lab Protocol in Rabbit
		3.1 Stage 1: Superovulation and Oocyte Collection
		3.2 Stage 2: Enucleation of Oocytes
		3.3 Stage 3: Donor Cell Preparation
		3.4 Stage 4: Nuclear Transfer, Embryo Culture, and Transfer
		3.5 Stage 5: Characterization of Donor Cells
	4 Therapeutic Cloning (or Known as Biomedical Cloning)
	5 Ethical Considerations in Human Cloning
	6 Ethical Considerations in Animal Cloning
	7 Honolulu Technique of Cloning
	8 Recent Advances in Cloning
	9 A Case Study
	References
Chapter 5: Immunohistochemistry
	Points to Remember
	1 Principle
	2 History of Immunohistochemistry
	3 Outline of the Protocol of IHC
	4 Detailed Protocol of Immunohistochemistry
		4.1 IHC-Paraffin Protocol (IHC-P)
			4.1.1 Optimizing a New Antibody for IHC-P
				Antigen Retrieval
				Primary Antibody Concentration
				Detection
			4.1.2 Fixation
			4.1.3 Deparaffinization
		4.2 Materials and Reagents
		4.3 Method
	5 Buffer Solutions for Heat-Induced Epitope Retrieval
	6 Heat-Induced Epitope Retrieval Methods
		6.1 Method
		6.2 Notes and Tips
		6.3 Materials and Reagents
		6.4 Method
		6.5 Notes
		6.6 Materials and Reagents
		6.7 Method
		6.8 Enzymatic Antigen Retrieval
	7 Enzymatic Retrieval, Pipetting Method
		7.1 Materials and Reagents
		7.2 Method 1
	8 Enzymatic Retrieval, Immersion Method
		8.1 Materials and Reagents
		8.2 Method
	9 Immunohistochemical Staining
		9.1 General Guidelines
		9.2 Protocol
		9.3 Controls
		9.4 Notes and Tips
		9.5 Signal Amplification
	10 Application for Immunohistochemistry
		10.1 Immunohistochemistry for Growth Hormone in Cattle Pituitary: A Case Study
		10.2 Immunohistochemistry for CD4 in Sheep Uterus
		10.3 Immunohistochemistry for CD8 in Sheep Uterus
	References
Chapter 6: Western Blotting
	Points to Remember
	1 Introduction
	2 Principle
		2.1 Basic Steps for Western Blotting
	3 Protocol
		3.1 Sample Preparation
		3.2 Gel Electrophoresis: Gel Preparation
		3.3 Gel Electrophoresis: Sample Buffer
			3.3.1 Loading Samples and Running the Gel
	4 SDS
		4.1 Blotting
		4.2 Blotting Membranes
		4.3 Visualization of Proteins in Membranes: Ponceau Red Stain
		4.4 Blocking
		4.5 Antibody Probing
		4.6 Detection with Substrate
	5 Principles of the Procedure: A Case Study
	References
Chapter 7: Introduction to Bioinformatics
	1 Basic Principle
	2 Sections of Bioinformatics
	3 Proteomics
	4 Prediction of Secondary Structure
		4.1 PyMOL View (3 D Structural Analysis) of Protein
			4.1.1 I-mutant
		4.2 Provean software
		4.3 String
	5 Application of Bioinformatics with Suitable Examples
		5.1 Functional Assessment How Mutations in the Gene Effects Its Functional Properties: A Case with Cytochrome B Gene in Sheep
		5.2 Three-Dimensional Structure Prediction and Model Quality Assessment
		5.3 H-Bonding Pattern Analysis and Protein-Protein Interaction Network Depiction
		5.4 Prediction of Post-translational Modification Sites in Mutant Cytochrome B
	6 Genomics
	7 Structure Analysis
		7.1 Database
	8 Systems Biology
	9 Evolutionary Biology
	10 Population Genetics
	11 Transcriptomics
		11.1 Databases
	12 Biophysics
	13 Imaging
	References
Chapter 8: Molecular Evolutionary Study: Phylogenetic Tree
	1 Basic Principle
	2 Types of Phylogenetic Tree
		2.1 MAFFT
		2.2 MEGA (Molecular Evolutionary Genetics Analysis)
		2.3 Protocol
			2.3.1 Step 1: Acquiring the Sequences
				Step 1.1
				Step 1.2: Which BLAST Algorithm to Use?
				Step 1.3: DNA Sequences
				Step 1.4: Protein Sequences
				Step 1.5: Alternatives to MEGA5 for Identifying and Acquiring Sequences
			2.3.2 Step 2: Aligning the Sequences
				Step 2.1
				Step 2.2
				Step 2.3
				Step 2.4
				Step 2.5: An Alternative to Aligning with MEGA5
			2.3.3 Step 3: Estimate the Tree
				Step 3.1
				Step 3.2
				Step 3.3
				Step 3.4
				Step 3.5: Estimating the Reliability of the Tree
				Step 3.6: Alternatives to MEGA5 for Estimating the Tree
			2.3.4 Step 4: Present the Tree
				Step 4.1
				Step 4.2
				Step 4.3
				Step 4.4
				Step 4.5: Publishing the Tree
				Step 4.6: Alternatives to Drawing Trees Within MEGA5
		2.4 Using MEGA5 on Macintosh Computers
		2.5 MEGA: A Case Study
		2.6 PHYLIP
		2.7 Reduced Median Network Theory: A Case
		2.8 Neighbor Joining Tree
	3 Effective Population Size
		3.1 How to Revive an Extinct Animal
	References
Chapter 9: Real-Time or Quantitative PCR
	1 Principles
	2 Protocol
	3 Common Parameters of Primer Design
		3.1 Primer Length
		3.2 Primer Melting Temperature (Tm)
		3.3 Annealing Temperature
		3.4 Product Size
		3.5 Mg2+ Concentration
		3.6 Repeats
		3.7 Runs
		3.8 3′ Stability
		3.9 GC Clamp
		3.10 Graphs: Melt Curve ABI
	4 Basics of Real-Time PCR
		4.1 Introduction
		4.2 The Advantages of Real-Time PCR
		4.3 Overview of Real-Time PCR
		4.4 Real-Time PCR Steps
		4.5 Real-Time PCR May Be Grouped as of Two Types
		4.6 Overview of Real-Time PCR Components
	5 Good Experimental Technique
	6 Real-Time PCR Primer Design
		6.1 Primer Design Software
	7 Real-Time PCR Analysis Technology
		7.1 Threshold
			7.1.1 Ct (Threshold Cycle)
		7.2 Melting Curve (Dissociation Curve)
	8 Real-Time PCR Fluorescence Detection Systems
		8.1 Real-Time Fluorescent PCR Chemistries
		8.2 5′ Nuclease Assay Specificity
		8.3 SYBR Green Assay Specificity
		8.4 SYBR Green Dye Dissociation
			8.4.1 Dye Differentiation
	9 Melting Curve Analysis
		9.1 Melting Curve Investigation and Detection Systems
		9.2 Significance of Melting Curve Analysis
			9.2.1 Melting Curve Analysis and Primer-Dimers
		9.3 How to Perform Melting Curve Analysis
		9.4 Passive Reference Dyes
	10 Contamination Prevention
		10.1 Uracil N-Glycosylase (UNG)
		10.2 How UNG Carryover Hindrance Works
	11 Multiplex Real-Time PCR
		11.1 Introduction to Multiplexing
		11.2 Multiplexing Benefits
		11.3 Instrumentation for Multiplexing
		11.4 Chemistry Recommendations for Multiplexing
		11.5 Dye Choices for Multiplexing
		11.6 Multiplex PCR Saturation
			11.6.1 Duplex Scenario 1
			11.6.2 Duplex Scenario 2
			11.6.3 Duplex Scenario 3
		11.7 Multiplex Primer Interactions
		11.8 Internal Controls and Reference Genes
		11.9 Relative Gene Expression Analysis Using Housekeeping Genes
	12 Real-Time PCR Instrument Calibration
		12.1 Excitation/Emission Difference Corrections
		12.2 Universal Optical Fluctuations
		12.3 Precision Improvement
		12.4 Atypical Optical Fluctuations
	13 Application for Real-Time PCR
	14 Evaluation of TaqMan qPCR System Integrating Two Identically Labeled Hydrolysis Probes in Single Assay: A Case Study
	References
Chapter 10: Tissue or Cell Cultures
	1 Principle
	2 History of Tissue Culture
	3 Fibroblast Subculturing Protocol
	4 Protocol for Lymphocyte Culture
	5 Techniques of Animal Cell Culture
	6 Some Characteristics of Animal Cell Growth in Culture
	7 Primary Cell Cultures
	8 Secondary Cell Cultures and Cell Lines
	9 Type of Cell Lines
	10 Environmental Factors Required for Culturing the Animal Cells
	11 Cryopreservation of Animal Cells
	12 Equipment Required for Animal Cell Culture
	13 Uses of Cell Culture
	References
Chapter 11: DNA Microarray
	1 Basic Principles
	2 Brief Overview of the Protocol
		2.1 Suppression of Noise
	3 DNA Microarray Protocol in Brief
	4 Detail Protocol of DNA Microarray
		4.1 Analysis
	5 Protocol For Preparing Poly-l-Lysine Slides for Microarrays
		5.1 Detail Protocol
	6 Protocol for Amplifying Products to Print on Array
	7 Protocol for Cleanup and 384-Well Arraying of PCR Products
	8 Protocol for Post-processing Microarrays
		8.1 Hydration/Heat Fixing
		8.2 Surface Blocking
	9 Protocol for Total RNA Isolation
	10 Protocol for Reverse Transcription and Amino-Allyl Coupling
	11 Protocol for Array Hybridization
	12 Protocol for Array Washing
	13 Uses of DNA Microarray
	14 The Birth of the Modern DNA Array
		14.1 Applications of Microarrays
			14.1.1 Gene Expression Analysis
		14.2 Gene Expression Analysis via Microarrays
			14.2.1 Transcription Factor Binding Analysis
		14.3 Genotyping
		14.4 Data Standards and Data Exchange
	15 Limitations of DNA Microarrays
	16 The Future of DNA Arrays
	References
Chapter 12: Epigenetics and DNA Methylation
	1 Principles
	2 Protocol for Studying Epigenetics
		2.1 Principles
	3 Summary of Epigenetic Gene Regulation
	4 DNA Methylation
		4.1 5-Methyl Cytosine Is Found in Heterochromatic Regions
	5 DNA Methylation
		5.1 Protocols
			5.1.1 Bisulfite Modification (Conversion) of DNA
		5.2 Materials and Reagents
		5.3 Procedure
	6 Bisulfite Modification for Nanogram Quantities of DNA
		6.1 Notes
	7 Bisulfite Genomic DNA Sequencing
		7.1 Bisulfite Sequencing of Small DNA/Cell Samples (Epigenome Network of Excellence)
		7.2 Bisulfite Sequencing of Very small Samples (Epigenome Network of Excellence)
		7.3 DNA Methylation
			7.3.1 A. CpG Islands
			7.3.2 DNA Methylases
		7.4 Techniques to Assess DNA Methylation
			7.4.1 Methylation-Sensitive Restriction Enzymes
		7.5 Bisulfite Sequencing
			7.5.1 Luciferase-Based Sensors of DNA Methylation
	8 Histone Modification
		8.1 Histone Modification and Histone Variants
			8.1.1 Histone Acetylation and Deacetylation
		8.2 Protocol to Determine HDAC Inhibitor Potency Using the HDAC-Glo I/II Assays and Cell Extracts or Purified Enzyme
			8.2.1 Materials Required
			8.2.2 Histone Methylation
			8.2.3 Histone Phosphorylation
		8.3 Protocol to Determine CDK1 Inhibitor Potency Using the ADP-Glo Kinase Assay + CDK1/CyclinA2 Kinase Enzyme System
			8.3.1 Materials Required
			8.3.2 Reagent Preparation
		8.4 Histone Ubiquitination
		8.5 Sumoylation as a Mechanism of Epigenetic Regulation
			8.5.1 Interpreting DNA Methylation and the Histone Code
			8.5.2 Noncoding RNAs
	9 Epigenetic Inheritance
	10 Epigenetics and Disease
	11 Gene Imprinting
		11.1 Higher-Order Chromatin Structures
		11.2 Noncoding RNA, Antisense RNA, and RNA Interference
		11.3 Imprinting in Mammals
		11.4 DNA Methylation and Igf2-H19 Imprinting in Mammals
		11.5 Chromatin Domains and the CTCF Insulator
			11.5.1 Antisense Transcripts and Mammalian Imprinting
	12 Epigenome Writers
		12.1 Writers, Readers, and Erasers of Epigenetic Marks
	13 Transcription Regulator
	References
Chapter 13: RNA Sequencing (RNA-seq)
	1 Basic Principle
	2 Introduction to RNA Sequencing
		2.1 Basic Mechanism of Action of RNA-seq
		2.2 An RNA-seq Protocol
			2.2.1 Experiment Planning
		2.3 cDNA Library Preparation
		2.4 cDNA Sequencing
		2.5 RNA-seq Data Analysis
		2.6 Featured Workflow for RNA-seq Analysis
			2.6.1 Library Preparation
			2.6.2 Sequencing
				Production Scale Sequencers
		2.7 Data Analysis
			2.7.1 Intuitive Analysis of RNA-seq Data
			2.7.2 Benefits of RNA-seq Data Analysis with BaseSpace Apps
		2.8 Data Analysis
			2.8.1 Interpretation of RNA-seq Data
				TopHat Alignment
					Illumina, Inc.
				App Highlights
					Current Limitations
					Reference Documents
				App Highlights
	3 RNA-seq in Details
		3.1 Applications of RNA-seq
	4 Protocol for RNA-seq Experiment
		4.1 Preparation of Total RNA
		4.2 Library Preparation
			4.2.1 RNA Fragmentation
			4.2.2 Reverse Transcription
			4.2.3 Adapter Ligation
			4.2.4 Library Cleanup and Amplification
			4.2.5 Library Quantification, Quality Control and Sequencing
		4.3 Data Analysis
		4.4 Experimental Design
	5 RNA-seq Technologies
		5.1 Illumina
		5.2 Ion Torrent
		5.3 SOLiD
	6 Non-coding RNA-seq
	7 Single-Cell RNA-seq Indexing/Barcoding
		7.1 Gene Quantification and Differential Expression Testing
		7.2 Data Visualization and Higher Level Analysis
	8 Emerging Technologies
		8.1 Automatic Data Analysis Workflows
	9 Pipelines for the Identification of Alternative Splicing and RNA Editing Sites
	10 Pipelines for Detection of Fusion Transcripts
	11 Pipelines for the Analysis of Small, Non-coding RNAs
	12 Tools for Single-Cell RNA Profiling Experiments
	13 Single-Cell RNA Sequencing: Sample Preparation
	14 Single-Cell Isolation Methods for scRNA-seq
		14.1 Limiting Dilution
		14.2 Micromanipulation or Manual Cell Picking
		14.3 Fluorescence-Activated Cell Sorting or FACS
		14.4 Laser-Assisted Tissue Microdissection
		14.5 Microfluidics
		14.6 Library Preparation for scRNA-seq
		14.7 Analysis of DATA After NGS
		14.8 Other Applications
	15 Summary
	References
Chapter 14: Next-Generation Sequencing
	1 Basic Principle
	2 A Brief History or Genesis of Next-Generation Sequencing
	3 Basic Workflow for NGS Experiments
	4 Template Generation
	5 Commonly Used NGS Platforms
	6 Applications for NGS
	7 DNA-Protein Interaction
	8 Detection of Non-coding RNA
	9 Mutation Discovery
	References
Chapter 15: Metagenomics
	1 Basic Principles
	2 Metagenomics Is an Important Field of Current Study with Many Practical Outcomes
	3 A Case Study with Classical Example in Case of Bone and Joint Infection Depicting the Need of Clinical Metagenomics as an Im...
		3.1 Material and Methods
			3.1.1 Samples
		3.2 DNA Manipulations
		3.3 Bioinformatic Methods
		3.4 Identification of Multiple Clones Within Species
	4 Metagenomic Analysis for Detection of Probiotics (Bacterial) for Growth, Feed Conversion Efficiency, Metabolism and Immunity
		4.1 Protocol
	5 Metagenomic Analysis for Detection of Viral Diversity
		5.1 Protocol in Brief
	6 Phylogenetic Analysis
	7 Metagenomic Work May Be Described in the Following Layout
	8 Detailed Protocol for 16S Metagenomic Sequencing Library Preparation
	9 Workflow Summary
	10 Analyze on MSR or BaseSpace
	11 Amplicon Primers
	12 Amplicon PCR
		12.1 Procedure
	13 PCR Clean-Up
		13.1 Preparation
		13.2 Procedure
	14 Index PCR
		14.1 Consumables
		14.2 Procedure
	15 PCR Clean Up 2
		15.1 Consumables
		15.2 Procedure
	16 Validation of Library
	17 Library Quantification, Normalization, and Pooling
	18 Library Denaturing and MiSeq Sample Loading
		18.1 Consumables
		18.2 Preparation
		18.3 Denaturation of DNA
		18.4 Dilution of Denatured DNA
		18.5 Denaturation and Dilution of PhiX Control
		18.6 Combine Amplicon Library and PhiX Control
	19 MiSeq Reporter Metagenomics Workflow
		19.1 Consumables and Equipment
	20 Applications of Metagenomics
	References
Chapter 16: Genome-Wide Association Studies/SNP Chips
	1 Basic Principle
	2 Data Analysis for GWAS
	3 Precomputed Summary Statistics Utilizing Standard Single-Marker Statistical Analysis Methods for GWAS
		3.1 Raw Data or Genotype-Level Data
		3.2 Raw Data Processing
		3.3 Data Annotation
			3.3.1 Study Annotation Comprises
			3.3.2 Sample Annotation
			3.3.3 Platform Annotation
			3.3.4 Exclusion Lists
	4 Data Exploration and Generation of Summary Statistics
		4.1 QC Filtering
		4.2 Stratification Analysis
		4.3 Multi-Dimensional Scaling (Fig. 4)
	5 Genomic Control
		5.1 Association Testing
			5.1.1 Population-Based Association Testing
		5.2 Quantitative Analysis
		5.3 Family-Based Association Testing
		5.4 Adjusted Test Statistics
		5.5 Constructing a Bioset
			5.5.1 From Pre-computed Analyses and Analyses of Genotype-Level or Raw Data
	6 GWAS Studies in General Consist of Discovery and Replication Phases
		6.1 Imputation
		6.2 Tagging Biosets with Ontology Terms
		6.3 Ranking of SNVs
	7 Genome-Wide Association Studies for Candidate Genes for Growth Relevant Traits in Pigs: A Case Study
		7.1 Animals and Phenotypes
		7.2 Genotyping, Imputation, and Quality Control
		7.3 Estimation of Genetic Parameters
		7.4 Genome-Wide Association Study
		7.5 Variance Explained by Candidate Regions
		7.6 Identification of Candidate Genes
	8 Another Case Study: Genome-Wide Association Studies for Candidate Genes for Growth Relevant Traits in Pigs
		8.1 Animals and Traits
	9 Study Design: Case Study
	10 Quality Control (QC) in GWAS
		10.1 QC for Individuals (Including Population Stratification)
		10.2 QC for SNPs
		10.3 SNP Selection for Replication I Stage
		10.4 Genotyping in Replication Stages and Platform Validation
		10.5 SNP Selection for Replication II Stage
	11 QC in Replication Studies
	12 Statistical Analysis
	13 Imputation and Regional Association Plot
	14 Fine Mapping
	15 Additional Check for Genotyping Quality
	16 Expression Quantitative Trait Loci (eQTL) Analysis
	References
Chapter 17: Exome Sequencing
	1 Principle
	2 Protocol
		2.1 Outline
	3 Protocol
	4 Sequencing
	5 Exome Kit Descriptions and Protocol Overview
		5.1 Infinium Omni5Exome-4 Kit (Illumina)
		5.2 Infinium OmniExpressExome-8 Kit
		5.3 Infinium Exome-24 Kit Arrays
		5.4 Infinium Omni2.5Exome-8 Kit
		5.5 Agilent HaloPlex
			5.5.1 Protocol Overview
		5.6 Agilent SureSelect
			5.6.1 Protocol Overview
		5.7 Agilent SureSelect QXT
		5.8 IDT xGEN Exome
			5.8.1 Protocol Overview
		5.9 Illumina Nextera Rapid Capture Exome
			5.9.1 Protocol Overview
		5.10 Illumina TruSeq Exome
			5.10.1 Protocol Overview
		5.11 Roche Nimblegen SeqCap
			5.11.1 Protocol Overview
		5.12 MYcroarray MYbaits
			5.12.1 Protocol Overview
	6 Factors Affecting Capture Efficiency
	7 Calculation of the Amount of Sequencing Needed for Exome Analysis Study: Calculate On-Target Rate or Enrichment Efficiency
	8 Calculation of Mean Normalized Coverage
	9 Exome Sequencing Data Requirement
	10 Determination of the Amount of Sequencing Needed to Meet Coverage Requirement
	11 Recommended Read Length for Exome Study
	12 Basic Recommendations for Calling Variants and Analyzing Depth of Coverage
		12.1 Exome Variant Analysis Recommendations
		12.2 Exome Coverage Analysis Recommendations
	13 Off-Target Reads (Reads Which Are Not Aligning to the Target Region)
	14 Considerations for Whole Exome Sequencing
		14.1 Sequencing Instrument and Read Length for Exome-seq
		14.2 Sequencing Coverage for Exome Sequencing
		14.3 Sequencing Coverage or Depth Required for Whole Exome Sequencing
		14.4 Choice of Exome Sequencing Capture Kit
		14.5 Comparison of the Annotation and Exome Capture Design Between Each Kit
		14.6 Choice of Whole Genome Sequencing (WGS) or Whole Exome Sequencing (WES)
	15 Exome Sequencing Categories
	16 Let Us Discuss in Details Exome Sequencing Practical Concern
		16.1 Softwares Employed for Data Analysis for Exome Sequencing
		16.2 Applications of Exome Sequencing
		16.3 Application of Exome Sequencing in Animal Science
		16.4 Exome Sequencing
		16.5 Advantages of Exome Sequencing
	17 Practical Application
		17.1 Exome Sequencing in Human
		17.2 Comparison for Exome Sequencing with Whole Genome Sequencing
		17.3 Challenges
		17.4 Data Analysis
		17.5 The Pipeline for Exome Sequencing Includes Three Important Phases as Depicted in Fig. 7
		17.6 Bioinformatics Issues
	18 Laboratory Protocol: A Case Study-A Protocol for Whole-Exome Sequencing in Newborns with Congenital Deafness-A Prospective ...
		18.1 Whole-Exome Sequencing
		18.2 Bioinformatic Analysis and Variant Filtering
		18.3 Variant Interpretation
	19 Evaluation Survey
		19.1 Analysis of Data
	20 Another Case Study for Exome Sequencing: X-exome Sequencing of 405 Unresolved Families Identifies Seven Novel Intellectual ...
		20.1 Subjects
		20.2 Methods
	21 Morphological Studies of Mouse Hippocampal Neurons
	22 Other Important Analyses
		22.1 Genome Wide Pathway Analysis (GWPA)
		22.2 Candidate Pathway Analysis
	References
Chapter 18: RNA Interference
	1 Basic Principle
		1.1 Brief History of RNA Interference
		1.2 Protocol
	2 Materials
		2.1 Making shRNA Constructs
		2.2 Making miRNA Constructs
		2.3 Cell Culture and Transfection
		2.4 Western Blot
		2.5 Northern Blot
		2.6 Reverse Transcription and Quantitative PCR
	3 Methods
		3.1 Design of siRNA, shRNA, and miRNA
			3.1.1 Background
			3.1.2 Designing siRNA
			3.1.3 Designing shRNA
			3.1.4 Designing miRNA
		3.2 Making shRNA Constructs
		3.3 Making miRNA Constructs
		3.4 Testing siRNA, shRNA, and miRNA Constructs
			3.4.1 Transfecting Cells
			3.4.2 Determination of Protein Knockdown Using Western Blot
			3.4.3 Determining RNA Knockdown Using Northern Blot
			3.4.4 Determining RNA Knockdown Using Reverse Transcription and Quantitative PCR (RT-qPCR)
				RNA Interference Has a Wide Range of Applications
				Other Forms of RNA Interference
					Piwi-Interacting (pi) RNAs
					Short-Hairpin (sh) RNAs
					Small Modulatory (sm)RNAs
	4 Making RNAi Work: Successful Transfection
		4.1 In Vitro Transfection Mechanisms and Protocols Naked Delivery
		4.2 Chemical Transfection
		4.3 Vector-Mediated Delivery: Overcoming Transient Silencing
		4.4 In Vivo Transfection
		4.5 Naked siRNA
		4.6 Hydrodynamic Delivery of siRNA
		4.7 Chemical Modifications Enhancing Transfection In Vivo
	5 Applications of RNA Interference in Pathology
		5.1 RNAi in Mechanistic Pathology
		5.2 RNAi in Animal Models of Disease
		5.3 RNAi as a Therapeutic Agent
	6 Conclusion
	References
Chapter 19: Expressing Cloned Genes for Protein Production, Purification, and Analysis-Biopharming
	1 Basic Principle
		1.1 Study for Protein Shape and Function
		1.2 Study of the Impact of Proteins on Genes or Other Proteins
	2 Expression and Purification of Recombinant Protein in Bacteria and Yeast
		2.1 Basic Steps to Obtain Recombinant Protein
	3 Features of Host: Prokaryotic Systems, Example-E. coli
	4 Features of Eukaryotic Systems
	5 Applications of Genetic Verification and Detection of Open Reading Frame (ORF) Expression
		5.1 Functional Analyses: Enzymatic Activity Analysis
		5.2 Post-translational Modification Analysis
		5.3 Molecular Interaction Detection Protein-Protein
		5.4 Protein-DNA and Protein-RNA
		5.5 DNA-RNA and RNA-RNA
		5.6 Molecular Structure and Localization Analyses: Characterization of Membrane Association
		5.7 Non-natural Amino Acid Incorporation
		5.8 Protein Folding and Chaperonin Interactions
		5.9 Real-Time Translation/Folding Assays
	6 Macromolecular Assembly
		6.1 Molecular Structure Analysis
	7 Molecular Diagnostics Protein Truncation Test
		7.1 High-Throughput Screening
			7.1.1 Screening for Viral-Specific Translation Inhibitory Compounds
		7.2 Identification of Novel Orphan Receptors
		7.3 Functional Genomics In Vitro Expression Cloning (IVEC)
		7.4 Ribosomal Display for Cell-Free Protein Evolution
		7.5 Preparative Synthesis: Large-Scale Protein Expression and Purification
		7.6 In Vitro Translation
	8 Cell-Free Expression Systems
		8.1 Two Approaches for In Vitro Protein Synthesis Based on the Starting Genetic Material: RNA or DNA
		8.2 Rabbit Reticulocyte Lysate
		8.3 E. coli Cell-Free System
		8.4 ``Linked´´ and ``Coupled´´ Transcription: Translation Systems
			8.4.1 Linked Transcription: Translation
		8.5 Human In Vitro Protein Expression System
	9 Important Elements for Translation
	10 Ribosomal Binding Site Sequence Requirements
	11 Metagenomics Is the Study of Genetic Material Recovered Directly from Environmental Samples
		11.1 Metatranscriptomics
		11.2 Practical Protocols for Production of Very High Yields of Recombinant Proteins Using Escherichia coli
			11.2.1 Molecular Cloning
			11.2.2 Double-Colony Selection
			11.2.3 Traditional IPTG-Induction Expression
			11.2.4 Autoinduction Expression
			11.2.5 High-Cell-Density IPTG-Induction Expression
			11.2.6 NMR Spectroscopy
		11.3 Selection of Appropriate Expression Vector Is Very Much Essential Because of Post-translational Modifications
		11.4 Cell Lysis and Protein Purification
		11.5 Mammalian Cell-Based Protein Expression
	12 Protein Expression Protocol and Troubleshooting
		12.1 Expression Protocol in E. coli
			12.1.1 Phase 1: Codon Optimized Gene Synthesis and Vector Construction
			12.1.2 Phase 2: Transform Expression Vector into E. coli Competent Cells
			12.1.3 Phase 3: Starter Culture
			12.1.4 Phase 4: Expansion of Starter Culture
			12.1.5 Phase 5: Induction
			12.1.6 Phase 6: Cells Collection and Lysis
		12.2 Expression Troubleshooting
			12.2.1 No/Low Expression
			12.2.2 Protein Aggregation
			12.2.3 Truncated Protein
			12.2.4 Protein Inactivity
	References
Chapter 20: Transgenesis and Biopharming
	1 Basic Principle
	2 Outline of the Protocol of Transgenesis Process (Transgenic Mouse)
	3 Transgenesis; Methodology
		3.1 Retrovirus-Mediated Gene Transfer
		3.2 DNA Microinjection Method
		3.3 Introduction of Genetically Engineered Embryonic Stem Cells into an Early Stage Developing Embryo Prior to Implantation in...
			3.3.1 Steps of the Procedure
				Considering the Above Facts, We Can Devise Certain Methods to Enrich DNA Integration at the Specific Sites
					A Procedure Called Positive/Negative Selection Is Implemented
			3.3.2 Possible Results
		3.4 Scientific and Medical Applications of the ES Cells Method of Transfection
			3.4.1 Chemical Transfection
				Calcium Phosphate Method
				Transfection with Polyplexes
				Transfection with Liposomes and Lipoplexes
			3.4.2 Physical Transfection
				Bacterial Vector
					Methodology
				Viral Vector
					Examples
				Nuclear Transfer Method (Nontransgenic Method)
					Applications of Nuclear Transfer
			3.4.3 Importance of Transgenesis
			3.4.4 Prospect for Transgenesis in Livestock Sector
			3.4.5 Some of the Common Terminologies in Transgenesis
	4 Ethics and Regulations for Preparation of Transgenic Product
		4.1 GEAC
		4.2 Patents and Rights
			4.2.1 Biopiracy
	5 Comparison of Transgenesis with Gene Editing
	References
Chapter 21: Rapid Amplification of cDNA Ends (RACE)
	1 Basic Principle
	2 Practical Protocol
	3 Summary of the 5′ RACE System
	4 Methods
		4.1 Components and Storage
		4.2 Additional Materials Required
		4.3 Performance and Limitations of Procedures
	5 Detail Methodology
		5.1 Isolation of Total RNA
		5.2 Design of the Gene-Specific Primers
		5.3 Primers for PCR (GSP2 and GSP3)
		5.4 Primer for First-Strand cDNA Synthesis (GSP1)
		5.5 Primers for Subsequent Cloning
		5.6 1x Wash Buffer for S.N.A.P. Procedure
		5.7 70% Ethanol Wash for S.N.A.P. Procedure
		5.8 First-Strand cDNA Synthesis
		5.9 S.N.A.P. Column Purification of cDNA
		5.10 TdT Tailing of cDNA
		5.11 PCR of dC-Tailed cDNA
		5.12 Nested Amplification
	6 Interpretation of Results
		6.1 Analysis of 5′ RACE Results
		6.2 5′ RACE Controls
		6.3 Nested Amplification
	7 Troubleshooting Guide
		7.1 Testing the 5′ RACE System Using the Control RNA and DNA
		7.2 Conversion of First-Strand cDNA and Recovery of cDNA After S.N.A.P.
		7.3 Sequences of the Control Primers
		7.4 Control First-Strand cDNA Synthesis
		7.5 S.N.A.P. Column Purification of the Control cDNA
		7.6 TdT Tailing of the Control First-Strand cDNA
		7.7 PCR of cDNA, Tailed cDNA, and Control DNA
		7.8 General Troubleshooting Guidelines for the 5′ RACE System
		7.9 Minimizing RNase Contamination
		7.10 Tm Values for 5′ RACE and Control Primers
	References
Chapter 22: Gene Therapy
	1 Principle
	2 History of Gene Therapy
	3 Protocol
		3.1 Mode of Action
	4 Types of Gene Therapy
		4.1 Gene Augmentation
		4.2 Gene Replacement
	5 Specific Inhibition of Gene Expression
		5.1 siRNA Therapy
		5.2 Ribozymes
		5.3 Targeted Cell Death
	6 Cationic Liposome-Mediated Gene Transfer to Tumor Cells In Vitro and In Vivo
	7 Methods for the Use of Cytokine Gene-Modified Tumor Cells in Immunotherapy of Cancer-A Case Study
	8 Methods for Generation of Genetically Modified Fibroblasts for Immunotherapy of Cancer
	9 Methods for Gene Transfer to Synovium
	10 Methods for Adenovirus-Mediated Gene Transfer to Synovium In Vivo
	11 Methods for the Use of Stromal Cells for Therapeutic Gene Therapy
	12 Suppression of the Human Carcinoma Phenotype by an Antioncogene Ribozyme
	13 Methods for Cancer Gene Therapy Using Tumor Suppressor Genes
	14 Intramammary Expression and Therapeutic Effect of a Human Lysozyme-Expressing Vector for Treating Bovine Mastitis: A Case S...
	15 Let us Discuss the Detailed Protocol
		15.1 Materials and Methods
			15.1.1 Vector Construction
			15.1.2 Vector Preparation
			15.1.3 Vector Injection
			15.1.4 Enzymatic Assay
			15.1.5 Antibody Assay
			15.1.6 Western Blotting
			15.1.7 Bacteriological Test
			15.1.8 Cure Definitions
	16 Result
		16.1 Vector Preparation
		16.2 Expression of p215C3LYZ After Primary Injection by Acupuncture
		16.3 Expression of p215C3LYZ After Primary Intracisternal Injection
		16.4 Dose-Dependent Expression of p215C3LYZ in Mammary Gland
		16.5 Expression of p215C3LYZ Vector After Secondary Injection
		16.6 Detection of hLYZ by Western Blotting
		16.7 Antibody Response After p215C3LYZ Injection
		16.8 Therapeutic Effect of Vector Injection on Bovine Mastitis
	References
Chapter 23: Nutrigenomics
	1 Basic Principles
	2 Protocol
		2.1 Using a Genome-Wide Analysis of DNA Methylation
	3 Impact and Application of Nutrigenomics
		3.1 Food Industry
		3.2 Nutrient Uptake and Assimilation
		3.3 Nutrigenomics in Diseases
		3.4 Impact and Applications of Nutrigenomics in Animal Sector
	4 Future Scope of Nutrigenomics
	References
Chapter 24: Gene Editing
	1 Basic Technique
	2 Advantages of Gene Editing over Transgenesis
		2.1 Zinc Finger Technology (ZFN)
			2.1.1 DNA-Binding Domain
			2.1.2 DNA-Cleavage Domain
		2.2 TALEN Technology
		2.3 CRISPR-Cas Technology
	3 History of CRISPR-Cas Technology
	4 Outline for Gene Editing for ``Knock in´´ of Promising Gene
		4.1 Methodology for Step 1
		4.2 Methodology for Step 2
	5 Protocol for Gene Editing
		5.1 Gene Cloning and gRNA Design
		5.2 sCell Culture and Transfection
		5.3 Luciferase-SSA Assay, T7 Endonuclease I Assay (T7E1) and TA Clone Sequence
		5.4 In Vitro Transcription of gRNA and Cleavage Activity
		5.5 In Vitro Induction Experiment
			5.5.1 Isolation and Culture of Chicken ESCs
			5.5.2 In Vitro Induction
		5.6 Microinjection of the Cas9/gRNA Plasmid into Chicken Embryos
			5.6.1 Quantitative Real-Time PCR (qRT-PCR)
			5.6.2 Immunofluorescence
			5.6.3 FACS
		5.7 Data Analysis
	6 Protocol for CRISPR/Cas9-Based Knock-In Using the VIKING Method
		6.1 Reagents
			6.1.1 Cell Culture Reagents
			6.1.2 Transfection
			6.1.3 Plasmids
			6.1.4 DNA Extraction
			6.1.5 Genotyping
			6.1.6 Equipment
	7 Procedure
		7.1 Protocol I: Procedure for Knock-in/Knock-out in Cultured Cells Using the VIKING Method
			7.1.1 Day 1: Cell Culture
			7.1.2 Day 2: Transfection (Fig. 1a)
			7.1.3 Day 4: Selection
			7.1.4 Day 14: Clone Picking (Fig. 1b)
			7.1.5 Day 16: DNA Extraction
			7.1.6 Day 17: Genotyping (Confirmation of Knock-in by PCR)
		7.2 Genotyping by PCR
	8 Protocol II: Procedure for Knock-in in Cultured Cells Using the VIKING Method (Fig. 2)
		8.1 Day 1-16
		8.2 Day 17: Genotyping
		8.3 Genotyping by PCR
		8.4 Remarks
	9 Easi-CRISPR Protocol for Creating Knock-in and Conditional Knockout Mouse Models Using Long ssDNA Donors
		9.1 Application for CRISPR Technology
		9.2 Applications of CRISPR Genome Editing
			9.2.1 Types of CRISPR-Enabled Genome Edits
			9.2.2 Methods to Check Genome Editing On-Target Efficiency
			9.2.3 Confirming CRISPR Knockouts and Other Edits
		9.3 CRISPR off-Target Analysis with NGS
			9.3.1 Publicly Available Analysis Tools to Predict Off-Target Effects
			9.3.2 Unbiased Methods to Analyze Off-Target Effects
			9.3.3 More Ways to Use NGS in Genome Editing
			9.3.4 CRISPR-Cas9 Knockdown
			9.3.5 Advantages of CRISPR-Cas9 Knock Down
			9.3.6 CRISPR-Cas9
	References
Chapter 25: Introduction to Stem Cell Biology
	1 Basic Principle
	2 History of Stem Cell Biology
	3 Molecular Marker for Stem Cell
	4 Basic Pluripotent Stem Cell Culture Protocol
		4.1 Outline of the Protocol
	5 A Stem Cell Culture Considerations
		5.1 A1 Successful Stem Cell Culture
		5.2 A2 Quality Control of Cell Cultures
			5.2.1 Cell Line Sterility
			5.2.2 Cell Line Authenticity
			5.2.3 Cell Line Stability
	6 B Reagent Preparation
		6.1 B1 Preparation of 0.1% Gelatin Solution
			6.1.1 Supplies
			6.1.2 Reagents
			6.1.3 Procedure
				Prepare 0.1% Gelatin Solution
		6.2 B2 Preparation of iMEF Culture Medium
			6.2.1 Supplies
			6.2.2 Reagents
			6.2.3 Procedure
				Preparation of iMEF Culture Media
		6.3 B3 preparation of Basic Fibroblast Growth Factor (B-FGF) Stock Solution
			6.3.1 Supplies
			6.3.2 Reagents
			6.3.3 Procedure
				Making of 0.1% Bovine Serum Albumin (BSA) Solution
				Preparation of 10 Ug/mL b-FGF Stock Solution
				Aliquots of b-FGF (10 μg/mL) Stock Solution
		6.4 B4 Making of Pluripotent Stem Cell Culture Medium
			6.4.1 Supplies
			6.4.2 Reagents
			6.4.3 Procedure
				Preparation of Culture Medium (CM) Preparation
		6.5 B5 Preparation of Collagenase Solution
			6.5.1 Supplies
			6.5.2 Reagents
			6.5.3 Procedure
	7 C Cell Culture
		7.1 C1 Gelatin Coating of Culture Plates
			7.1.1 Supplies
			7.1.2 Reagents
			7.1.3 Procedure
				Coat Culture Plates Using 0.1% Gelatin Solution
		7.2 C2 Thawing and Seeding of Freezing Inactivated Mouse Embryonic Fibroblasts (iMEFs)
			7.2.1 Supplies
			7.2.2 Reagents
			7.2.3 Procedure
				Prepare Gelatin Plate(S) in the Biosafety Cabinet
				Thaw iMEFs
				Seed iMEFs in 6-Well Plate
				Post Seeding
		7.3 C3 Thawing and Seeding, and Substitute of Medium for Pluripotent Stem Cells
			7.3.1 Supplies
			7.3.2 Reagents
				Monitor iMEFs under Microscope
				Thaw Pluripotent Stem Cells
				Seed Pluripotent Stem Cells in 6-Well Plates
				Post Seeding
		7.4 C4 Substitute of Pluripotent Stem Cell Culture Medium
			7.4.1 Supplies
			7.4.2 Reagents
			7.4.3 Procedure
				Monitor hPSC Growth
				Change Medium for hPSC Culture
		7.5 C5 Passaging of Pluripotent Stem Cells on New iMEF Plates
			7.5.1 Supplies
			7.5.2 Reagents
			7.5.3 Preparation
				Establish when and how to Passage (or Split) Pluripotent Stem Cells
				Determine How to Channel (or split) Pluripotent Stem Cells
				Determine How Much Medium and Solution Are Needed for this Process
				Evaluate and Prepare Fresh iMEF Plates
				Procedure-Pick to Ruin-Enzyme Harvest Cells (No More than Two Plates at a Time)
				Spin and Disrupt Colonies
				Addition of Cells to iMEF Plates (Processing No More than Three Plates at a Time)
				Procedure-Pick to Keep Prepare Sterile Bent Pasteur Glass
				Transfer Areas of Undifferentiated Cells to Fresh iMEF Plates (Pick to Keep)
	8 Stem cell culture-Requirements and protocol
		8.1 Supplies
			8.1.1 Reagents
			8.1.2 Procedure
				Determine when to Harvest Cells
				Prepare Cryovials
				Determine how Much Culture Medium and Collagenase Solution Is Needed for this Process
				Set up 2x Cryopreservation Media (or Freezing Medium, FM) in the Biosafety Cabinet
				Incubate Cells with Collagenase Solution (Make No More than Two Plates at a Time)
				Harvest Cells
				Freeze Cells
		8.2 Protocols
	9 Culturing Mesenchymal Stem Cells (MSCs) in 2% Reduced Serum Medium-a Case Study
		9.1 Materials Needed
			9.1.1 For Recovery Only of Cryopreserved MSCs
			9.1.2 For passaging and cryopreservation of cells
		9.2 Preparing Media and Materials
		9.3 Recovery of Cryopreserved MSCs
		9.4 Subculturing MSCs
		9.5 Cryopreservation of MSCs
	10 Culturing Human Mesenchymal Stem Cells
		10.1 Materials Needed
			10.1.1 For Either Culture and Isolation from Bone Marrow
			10.1.2 For Serum-Free, Xeno-Free Cultures
			10.1.3 For Serum-Free Cultures
			10.1.4 For Isolating MSCs from Bone Marrow
		10.2 Preparing Media and Materials
			10.2.1 StemPro MSC SFM XenoFree entire Medium for Serum-Free, Xeno-Free Cultures (500 mL of complete medium)
			10.2.2 Primary Isolation Medium: StemPro MSC SFM XenoFree whole Medium include 2.5% human AB Serum for Isolation of MSCs from ...
			10.2.3 StemPro MSC SFM CTS Complete Medium Lacking Serum Cultures (500 mL of Complete Medium)
			10.2.4 Coating Culture Vessels with CTS CELLstart Substrate
			10.2.5 Recovering Cryopreserved Human MSCs
			10.2.6 Isolating MSCs From Bone Marrow
			10.2.7 Subculturing MSCs
				General Guidelines
		10.3 Propagating MSCs
		10.4 Cryopreservation of MSCs.
	Expected Results
		Maintaining Trilineage Potential Under Serum-Free and Xeno-Free Conditions
		Morphology of hMSCs Developed Under Serum-Free and Xeno-Free Environments
	References
Chapter 26: Transcriptomics
	1 Basic Principle
	2 Protocol
	3 Isolation of RNA
	4 Expressed Sequence Tags
	5 Serial and Cap Analysis of Gene Expression (SAGE/CAGE)
		5.1 Summary of SAGE
	6 Microarrays
		6.1 Summary of DNA Microarrays
	7 Principles and Advances
	8 Methods
		8.1 RNA-Seq
			8.1.1 Summary of RNAseq
		8.2 Data Analysis
		8.3 Image Processing
			8.3.1 Microarray and Sequencing Flow Cell
	9 Quality Control
	10 Alignment
	11 Quantification
	12 Validation
	13 Applications
		13.1 Diagnostics and Disease Profiling
	References
Chapter 27: Development of Biomarker
	1 Basic Principle
	2 There Are Two Approaches for Biomarkers
		2.1 Definition of a Biomarker
		2.2 Diagnostic Use of Biomarkers
	3 Marker
	4 Markers
	5 Biomarkers for Human Used for Diagnosis Purpose
	6 Summary for Exosomes as Biomarker for Various Diseases
		6.1 Exosome as a Biomarker for Metabolic Diseases-A Case Study
		6.2 Exosome-Based Strategies for the Diagnosis of Tumors-A Case Study
		6.3 Exosomes: Novel Biomarkers for Neurodegenerative Diseases
		6.4 Exosome and Other Human Pathological Conditions
		6.5 Diagnosis of Prion Diseases
	7 Biomarkers for Animals to Be Used for Marker-Assisted Selection or Genomic Selection
	8 History of MAS
		8.1 Methodological Advantage of MAS
		8.2 Limitations of MAS
	9 Current Application of MAS in Livestock
	10 A Simple Example of Molecular Marker (RFLP)
	11 Conventional Techniques for Detection of Polymorphism
	12 Conclusion
	References
Chapter 28: Single-Cell Genomics
	1 Basic Principles
	2 Introduction to Single-Cell RNA Sequencing
	3 Single-Cell Genome (DNA) Sequencing
	4 Single-Cell DNA Methylome Sequencing-Epigenetic Study
	5 Applications
	6 Single-Cell RNA Sequencing (scRNA-Seq)
	7 Advantages of Single-Cell RNA-Seq
	8 The Chromium Single-Cell Gene Expression Solution
	9 A Case Study: Single-Cell Reconstruction of the Early Maternal-Fetal Interface in Humans
	10 A Case Study: Molecular Classification and Comparative Taxonomics of Foveal and Peripheral Cells in Primate Retina
	11 Acquired Cancer Resistance to Combination Immunotherapy from Transcriptional Loss of Class I HLA: A Case Study
	12 Applications for Single-Cell Genomics
	13 High- and Low-Throughput Methods
	References
Chapter 29: Big Data Analysis
	1 The Advantages of Analysis for Big Data
	2 Data Mining
	3 Data Format
	4 Basic PLINK Command
	5 QC of Genetic Data
	6 Data Simulation Using HapMap Data
	7 Overview of QC Steps
	8 Controlling for Population Stratification
	References
Index