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TitleLipoproteins and Cardiovascular Disease: Methods and Protocols
LanguageEnglish
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Total Pages385
Table of Contents
                            Preface
Contents
Contributors
Part I: RNA and Gene Expression
	Chapter 1: Cloning Full-Length Transcripts and Transcript Variants Using 5' and 3' RACE
		1 Introduction
		2 Materials
			2.1 First-Strand cDNA Synthesis Using the SMART RACE cDNA Amplification Kit
				2.1.1 Before Beginning
				2.1.2 First-Strand cDNA Synthesis
			2.2 RACE PCR Reactions [ 14 ]
			2.3 Cloning and Sequencing RACE Products
		3 Methods
			3.1 First-Strand cDNA Synthesis Using the SMART RACE cDNA Amplification Kit
				3.1.1 Before Beginning
				3.1.2 First-Strand cDNA Synthesis [ 14 ]
			3.2 RACE PCR Reactions [ 14 ]
			3.3 Cloning and Sequencing RACE Products
		4 Notes
		References
	Chapter 2: Monitoring Gene Expression: Quantitative Real-Time RT-PCR
		1 Introduction
		2 Materials
			2.1 Total RNA Extraction from Tissue Using TRIzol
				2.1.1 Isolation and Stabilization of Tissue
				2.1.2 RNA Extraction from Tissue (TRIzol)
			2.2 Isolation of RNA from Cultured Cells and RNA Cleanup Using the Qiagen RNeasy Mini Kit
			2.3 Quantification and Storage of Total RNA
			2.4 Reverse Transcription (RT)
			2.5 Real-Time PCR (qPCR)
		3 Methods 1
			3.1 Total RNA Extraction from Tissue Using TRIzol
				3.1.1 Isolation and Stabilization of Tissue
				3.1.2 RNA Extraction from Tissue (TRIzol)
			3.2 Total RNA Extraction from Cells and RNA Cleanup Using the Qiagen RNeasy Mini Kit
				3.2.1 For Cleanup of Total RNA from Tissue After TRIzol Extraction
				3.2.2 Cell Culture, Adherent Cells
			3.3 Quantification and Storage of Total RNA
			3.4 Reverse Transcription (RT)
			3.5 Real-Time PCR (qPCR)
				3.5.1 Program Setup
				3.5.2 cDNA Dilution
				3.5.3 Analysis
				3.5.4 Calculation of Relative Gene Expression Levels
		4 Notes
		References
	Chapter 3: Microarray Technology: Basic Methodology and Application in Clinical Research for Biomarker Discovery in Vascula...
		1 Introduction
		2 Materials
		3 Methods
			3.1 Study Design
			3.2 Sample Collection, Cell Lysis, and RNA Isolation, with Maintenance of RNA Integrity
				3.2.1 Whole Blood: Stabilization and Subsequent RNA Isolation Using the PAXgene™ Kit
				3.2.2 Purification of RNA with RNeasy Mini Kit: PBMCs, Cultured Cells, and Tissue Specimens
				3.2.3 Flow-Sorted Cells or Few Hundred Cells: Use of Ambion’s RNAqueous-Micro Kit for Isolating RNA from Specialized Cell P...
			3.3 Target-Labeled cRNA/cDNA Preparation
				3.3.1 First-Strand cDNA Synthesis (IVT Express Kit)
				3.3.2 Second-Strand cDNA Synthesis
				3.3.3 Cleanup of Double-Stranded cDNA [Sample Cleanup Module at Room Temperature (RT)]
				3.3.4 Synthesis of Biotin-Labeled cRNA by In Vitro Transcription (IVT) Reaction
				3.3.5 Cleanup and Quantification of Biotin-Labeled cRNA (Sample Cleanup Module at RT)
				3.3.6 Target Preparation from Flow-Sorted Cells or Few Hundred Cells
				3.3.7 Fragmentation of Biotinylated cRNA
				3.3.8 Preparation of Labeled cDNA for Hybridization onto Gene ST and Exon ST Arrays
			3.4 Microarray Hybridization, Washing, Staining, and Scanning
				3.4.1 Preparation of Hybridization Cocktail
				3.4.2 Prehybridization of the Probe Array
				3.4.3 Hybridization of the Probe Array
				3.4.4 Fluidics Station Setup
				3.4.5 Probe Array Washing, Staining, and Scanning
			3.5 Microarray Data Analysis
				3.5.1 Checking Scanned Image and Converting Fluorescence Intensities to Numerical Values
				3.5.2 File Types
				3.5.3 Preliminary Data Analysis
		4 Notes
		References
	Chapter 4: Northern Analysis of Gene Expression
		1 Introduction
		2 Materials
			2.1 RNA Preparation
				2.1.1 Tissue Isolation and Stabilization
				2.1.2 RNA Isolation
				2.1.3 (Optional) PolyA+ RNA
				2.1.4 RNA Quantitation
			2.2 Electrophoresis
				2.2.1 Pouring the Agarose Gel
				2.2.2 Preparing Samples and Standards
				2.2.3 Running the Gel
				2.2.4 Viewing and Imaging the Gel
			2.3 Blotting and Cross-Linking
				2.3.1 Sponge Method
				2.3.2 TurboBlotter (Upwards Blotting)
				2.3.3 Cross-Linking
			2.4 Working with 32 P
			2.5 Preparing the Probe
				2.5.1 32 P-Labeling of the DNA Fragment
				2.5.2 Removing Unincorporated Nucleotides with ProbeQuant™ G-50 Micro Columns
			2.6 Prehybridization, Hybridization, and Washing
				2.6.1 Prehybridization
				2.6.2 Hybridization
				2.6.3 Washes
			2.7 Detection
				2.7.1 Film
				2.7.2 Storage Phosphor/PhosphorImager Technology
			2.8 Probe Stripping
		3 Methods
			3.1 RNA Preparation
				3.1.1 Tissue Isolation and Stabilization
				3.1.2 RNA Isolation
				3.1.3 PolyA+ RNA
				3.1.4 RNA Quantitation and Determination of Sample Volume
			3.2 Electrophoresis
				3.2.1 Pouring the Agarose Gel
				3.2.2 Preparing Samples and Standards
				3.2.3 Running the Gel
				3.2.4 Viewing and Imaging the Gel
			3.3 Blotting and Cross-Linking
				3.3.1 Sponge Method (Adapted from Zeta-Probe GT Instruction Manual, BioRad)
				3.3.2 TurboBlotter (Downwards Blotting)
				3.3.3 Cross-Linking
			3.4 Working with 32 P
			3.5 Preparing the Probe
				3.5.1 32 P-Labeling of the DNA Fragment
				3.5.2 Removing Unincorporated Nucleotides with ProbeQuant™ G-50 Micro Columns
			3.6 Prehybridization, Hybridization, and Washing
				3.6.1 Prehybridization
				3.6.2 Hybridization
				3.6.3 Washes
			3.7 Detection
				3.7.1 Film
				3.7.2 Storage Phosphor/PhosphorImager Technology
			3.8 Probe Stripping
		4 Notes
		References
	Chapter 5: Laser Capture Microdissection for Analysis of Macrophage Gene Expression from Atherosclerotic Lesions
		1 Introduction
		2 Materials
			2.1 Animals and Tissue Processing
			2.2 Histological Detection of Macrophage Foam Cells for LCM
			2.3 Laser Capture Microdissection and RNA Isolation
			2.4 Real-Time Quantitative RT-PCR
		3 Methods
			3.1 Animals and Tissue Processing
			3.2 CD68 Immunodetection of Macrophages
			3.3 Hematoxylin and Eosin Y (H&E) Stain
			3.4 Laser Capture Microdissection
			3.5 Analysis of Macrophage Foam Cell Gene Expression by Real-Time Quantitative RT-PCR
		4 Notes
		References
Part II: Sequencing
	Chapter 6: Sequencing PCR-Amplified DNA in Lipoprotein and Cardiovascular Disease Research
		1 Introduction
		2 Materials
			2.1 Genomic DNA
			2.2 PCR DNA Amplification Reactions in 96- or 384-Well Formats
			2.3 Agarose Gel Electrophoresis
			2.4 Exonuclease I/Shrimp Alkaline Phosphatase PCR Cleanup Reactions
			2.5 BigDye Terminator Sequencing Reactions
			2.6 Option 1: Purification of Sequencing Reactions by Sephadex Gel Filtration
			2.7 Option 2: Automated Dye Terminator Magnetic Bead Purification
			2.8 DNA Sequencer and Sequence Analysis
		3 Methods
			3.1 DNA Preparation (Again Notes 2 and 3)
			3.2 PCR Amplification from Genomic DNA
			3.3 TAE Agarose Gel Electrophoresis
			3.4 Exonuclease I/Shrimp Alkaline Phosphatase PCR Cleanup Reactions
			3.5 Option 1: BigDye Terminator Sequencing Reactions and Sephadex Purification
			3.6 Option 2: Diluted BigDye Terminator Sequencing Reactions and Magnetic Bead Purification
			3.7 Analysis of Sequencing Reactions on a Capillary DNA Sequencer
		4 Notes
		References
	Chapter 7: Introduction to Next-Generation Nucleic Acid Sequencing in Cardiovascular Disease Research
		1 Introduction
		2 Second-Generation Sequencing
			2.1 Roche 454 Life Sciences FLX Genome Sequencer ( http://www.454.com)
			2.2 Illumina Genome Analyzer II/Solexa ( http://www.Illumina.com)
			2.3 Applied Biosystems SOLiD™ 3.0 Sequencer ( http://solid.appliedbiosystems.com)
		3 Applications for Second-Generation Sequencing
		4 Future Technologies
			4.1 Helicos Biosciences ( http://www.helicosbio.com)
			4.2 Pacific Biosciences ( http://pacificbiosciences.com)
			4.3 Oxford Nanopore Technologies ( http://nanoporetech.com)
		5 Conclusion: Clinical Applications in Cardiovascular Diseases
		6 Notes
		References
Part III: Transgenic, Knockout, and Knockdown Methodologies
	Chapter 8: Strategies for Designing Transgenic DNA Constructs
		1 Introduction
		2 Major Challenges for Controlling Transgene Expression
		3 Commonly Used Transgenic Cloning Vectors
			3.1 Plasmid Vectors
			3.2 Bacterial Artificial Chromosomes (BACs) and Other Large Pieces of DNA
		4 Main Applications of Transgenic Technology
			4.1 Gene Overexpression
			4.2 Promoter Characterization and Cell Linage Markers
			4.3 Gene Knockdown by RNA Interference
			4.4 Comple- mentation and Mutation Mapping
			4.5 Double or Multiple Transgenes
			4.6 Site-Specific Recombinase and Conditional Transgenic Lines
			4.7 Inducible Transgene Expression
		References
	Chapter 9: Purification of Plasmid and BAC Transgenic DNA Constructs
		1 Introduction
		2 Materials
			2.1 Commercial Kits
			2.2 Equipment and Supplies
			2.3 Chemicals
		3 Methods
			3.1 General Guidelines and Cautions for Purifying Microinjection-Quality DNA
				3.1.1 DNA Integrity
				3.1.2 DNA Purity
				3.1.3 Particles
				3.1.4 DNA Quantity and Concentration
			3.2 Isolation of the Transgenic Fragment from the Plasmid Vector
				3.2.1 Procedure
			3.3 Purification of BAC DNA for Microinjection
				3.3.1 Procedure
		4 Notes
		References
	Chapter 10: Pronuclear Microinjection and Oviduct Transfer Procedures for Transgenic Mouse Production
		1 Introduction
		2 Materials
			2.1 Mice
			2.2 Equipment
				2.2.1 Microinjection Setup
				2.2.2 Pipette Puller and Injection Needles
				2.2.3 Stereo Microscope
				2.2.4 CO 2 Incubator
				2.2.5 Sterilizers
			2.3 Tools and Supplies
			2.4 Media and Chemicals
		3 Methods
			3.1 Zygote Collection
			3.2 Pronuclear Microinjection
			3.3 Oviduct Transfer ( See Note 9 for Timing of Embryo Transfer)
		4 Notes
		References
	Chapter 11: Genotyping of Transgenic Animals by Real-Time Quantitative PCR with TaqMan Probes
		1 Introduction
		2 Materials
			2.1 DNA Isolation
			2.2 Determination of DNA Concentration
			2.3 Primers and MGB TaqMan Probes
			2.4 Calibration Standards
			2.5 Preparing the Plan
			2.6 Genomic DNA Solutions
			Sec00119
			2.7 Real-Time qPCR
		3 Methods
			3.1 DNA Isolation
			3.2 Determination of DNA Concentration
			3.3 Designing Primers and TaqMan MGB Probes for qPCR
			3.4 Preparing Calibration Standards for qPCR
			3.5 Preparing a Plan for the qPCR Experiment
			3.6 Preparing DNA Solutions for qPCR
			3.7 Preparing the qPCR Working Reagent Mix
			3.8 Preparing the qPCR Plate and Running qPCR
			3.9 Analysis of the Results
		4 Notes
		References
	Chapter 12: Generation of General and Tissue-Specific Gene Knockout Mouse Models
		1 Introduction
		2 Materials
			2.1 Isolation of MEF
			2.2 ES Cell Growing, Transfection, and Selection
			2.3 DNA Extraction from ES Cells
			2.4 Blastocyst Isolation, Targeted ES Injection, and Transplantation
				2.4.1 Animal Setting
				2.4.2 Materials
			2.5 PLTP KO (Conventional) Mouse Preparation
				2.5.1 Construction of PLTP Gene Replacement Vector
				2.5.2 PLTP KO Mouse Preparation
			2.6 Serine Palmitoyltransferase subunit 2 Tissue-Specific Mouse Preparation
				2.6.1 Construction of “Sptlc2-Flox” Gene Replacement Vector
				2.6.2 Preparation of Sptlc2 Flox/Frt Mice
				2.6.3 Preparation of Sptlc2 D Neo Mice
				2.6.4 Preparation of Sptlc2-Flox Mice
			2.7 Preparation of a Liver-Specific Sptlc2 KO Mouse
				2.7.1 Adenovirus-Cre Method
				2.7.2 Pharmacological Induction of a Cre Transgene
				2.7.3 Albumin Promoter/Enhancer-Cre Transgenic Mice
			2.8 Intestine-Specific Gene KO Mice
			2.9 Preparation of Macrophage-Specific Gene KO Mice
				2.9.1 LysM-Cre Transgenic Mouse Approach (myeloid cell-specific)
				2.9.2 Bone Marrow Transplantation Approach (hematopoietic cell-specific)
		3 Methods
			3.1 Isolation of MEF
			3.2 ES Cell Growing, Transfection, and Selection
			3.3 DNA Extraction from ES Cells
			3.4 Blastocyst Isolation, Targeted ES Injection, and Transplantation
				3.4.1 Animal Setting
				3.4.2 Procedure
			3.5 PLTP KO (Conventional) Mouse Preparation
				3.5.1 Construction of PLTP Gene Replacement Vector
				3.5.2 PLTP KO Mouse Preparation
			3.6 Serine Palmitoyltransferase subunit 2 Tissue-Specific Mouse Preparation
				3.6.1 Construction of the Sptlc2-Flox Gene Replacement Vector
				3.6.2 Preparation of Sptlc2 Flox/Frt Mice
				3.6.3 Preparation of Sptlc2 D Neo Mice
				3.6.4 Preparation of Sptlc2-Flox Mice
			3.7 Preparation of a Liver-Specific Sptlc2 KO Mouse
				3.7.1 Liver-Specific Sptlc2 Inactivation by Injecting Adenovirus-Cre into Sptlc2-Flox Mice
				3.7.2 Liver-Specific Sptlc2 Inactivation by Pharmacological Induction of a Cre Transgene in Sptlc2-Flox Mice
				3.7.3 Liver-Specific Sptlc2 Inactivation by Crossing Sptlc2-Flox Mice with Albumin Promoter/Enhancer-Cre Transgenic Mice
			3.8 Intestine-Specific Sptlc2 Inactivation by Crossing Sptlc2-Flox Mice with Villin 1-Cre Transgenic Mice
			3.9 Preparation of Macrophage-Specific Gene KO Mice
				3.9.1 Myeloid-Specific Sptlc2 Inactivation by Crossing Sptlc2-Flox Mice with LysM-Cre Transgenic Mice
				3.9.2 Bone Marrow Transplantation from Whole-Body Knockout Mice to a Acceptor Mice to Prepare Hematopoietic Cell-specific G...
		4 Notes
		References
	Chapter 13: Adeno-associated Viruses as Liver-Directed Gene Delivery Vehicles: Focus on Lipoprotein Metabolism
		1 Introduction
			1.1 Liver-Directed Gene Transfer
			1.2 Adenovirus
			1.3 Adeno-associated Virus
		2 Materials
			2.1 Transfection
			2.2 Purification
			2.3 Genome Copy Number Titration
			2.4 Endotoxin Assay
			2.5 Capsid Purity Assay
			2.6 Transduction of HepG2 Cells
			2.7 In Vivo Liver Transduction
		3 Methods
			3.1 Choosing the Vector
			3.2 Generating the Expression Cassette
			3.3 AAV Cis Plasmid Quality Control
			3.4 Production of Recombinant AAV by Transient Transfection Using HEK 293 Cells
				3.4.1 Preparation of HEK 293 Cells for Transfection
				3.4.2 Preparation of Transfection Cocktail
				3.4.3 Transfection of HEK 293 Cells
				3.4.4 Harvesting of Transfected Cells
				3.4.5 Storage of Cell Pellet/Suspension
			3.5 Purification of Recombinant AAV Vectors by Cesium Chloride Gradient Centrifugation
				3.5.1 Generation of Cell Lysate
				3.5.2 Preparation of First Cesium Chloride Gradient
				3.5.3 Fraction Collection After the First Centrifugation
				3.5.4 Determination of Refractive Index (RI)
				3.5.5 Preparation of Second Cesium Chloride Gradient
				3.5.6 Fraction Collection After the Second Centrifugation
				3.5.7 Desalting 
and Buffer Exchange
			3.6 Characterization of Purified AAV
			3.7 AAV Genome Copy Number Titration
				3.7.1 Cis Plasmid Standard Preparation
				3.7.2 Digestion of Un-encapsidated DNA
				3.7.3 Sample Preparation
				3.7.4 PCR Preparation
				3.7.5 Data Analysis
				3.7.6 Determination of Titer
			3.8 Assessment of Purity
			3.9 Endotoxin Analysis
				3.9.1 Sample Preparation (Biosafety Containment Hood)
				3.9.2 Kit Reagents Dilution and Standards Formulation
				3.9.3 Performing the Reaction
				3.9.4 OD Measurement and Data Interpretation
			3.10 Capsid Purity Assessment by SDS-PAGE
				3.10.1 Electrophoresis Apparatus Assembly and Gel Loading
				3.10.2 Gel Staining
				3.10.3 Gel Documentation and Densitometric Analysis
				3.10.4 Anticipated Results
			3.11 Verification of Transduction and Transgene Expression In Vitro
			3.12 Transduction of HepG2 Cells
				3.12.1 Growth of HepG2 Cells
				3.12.2 Seeding Plates with HepG2 Cells (Day 1)
				3.12.3 Infection with AAV Vector (Day 2)
				3.12.4 Harvesting Transduced Cells and Media (Day 5)
				3.12.5 Western Blotting
				3.12.6 Expected Results
				3.12.7 Alternative Means of Verifying Expression
			3.13 In Vivo Studies Using AAV Vectors to Target the Liver
			3.14 In Vivo Liver Transduction
		4 Notes
		References
	Chapter 14: Modulation of Lipoprotein Metabolism by Antisense Technology: Preclinical Drug Discovery Methodology
		1 Introduction
		2 Antisense Inhibitors to Lipoproteins
		3 Antisense Technology Preclinical Drug Discovery Process
			3.1 Identification of Lipoprotein-Related Targets
			3.2 Evaluation of ASOs In Silico
			3.3 Evaluation of ASOs In Vitro
			3.4 In Vivo Evaluation of ASO Leads
		4 Identification of Human Drug Candidates
		5 Conclusion
		References
Part IV: Special Topics
	Chapter 15: Chromatin Immunoprecipitation
		1 Introduction
		2 Materials
			2.1 Equipment
			2.2 Reagents
		3 Methods
			3.1 Before the Experiment
				3.1.1 Optimize Shearing with Your Sonicator
				3.1.2 Design Primers
				3.1.3 Obtain Antibodies
			3.2 Procedure for Chromatin Immunoprecipitation
				3.2.1 Cross-Link and Harvest Cells
				3.2.2 Lyse Cells and Shear Chromatin
				3.2.3 Immunopre-cipitation
				3.2.4 Input DNA Isolation
				3.2.5 DNA De-Crosslinking and Purification
				3.2.6 Assessment of Shearing
			3.3 Quantitative PCR Analysis
				3.3.1 Quantitative PCR
				3.3.2 Data Analysis
				3.3.3 Example
		4 Notes
		References
	Chapter 16: Measurement of Lecithin–Cholesterol Acyltransferase Activity with the Use of a Peptide-Proteoliposome Substrate
		1 Introduction
		2 Materials
			2.1 Reagents
			2.2 Reagent Setup
			2.3 Preparing Proteoliposome Substrate
			2.4 Preparing the LCAT Reagent Mix
		3 Methods
			3.1 LCAT Reaction
			3.2 Extracting the LCAT Reaction Products
			3.3 TLC Separation of CE from Cholesterol
			3.4 Calculations
		4 Notes
		References
	Chapter 17: Native–Native 2D Gel Electrophoresis for HDL Subpopulation Analysis
		1 Introduction
		2 Materials
			2.1 First Dimension
			2.2 Second Dimension
			2.3 Transfer
			2.4 Blocking, Antibody Incubation, and Detection
		3 Methods
			3.1 First Dimension
			3.2 Second Dimension
			3.3 Transfer
			3.4 Blocking, Antibody Incubation, and Detection
		4 Notes
		References
	Chapter 18: Western Blots
		1 Introduction
		2 Materials
			2.1 Gels
				2.1.1 SDS-PAGE
				2.1.2 1D Native Gels
			2.2 Transfer
			2.3 Blocking, Antibody Incubation, and Detection
		3 Methods
			3.1 Gels
				3.1.1 SDS-PAGE
				3.1.2 1D Native Gels
			3.2 Transfer
			3.3 Blocking, Antibody Incubation, and Detection
		4 Notes
		References
Appendix
	Working with RNA
Index
                        

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