Liquid Biopsies: Methods and Protocols (Methods in Molecular Biology, 2695)

Preface
Contents
Contributors
Chapter 1: The Protocol of Circulating Tumor Cell Detection
	1 Introduction
	2 Materials
		2.1 Reagents
		2.2 Equipment
	3 Procedures
		3.1 Enrichment by Negative Immunomagnetic Particle Assays
		3.2 FISH by Immunofluorescence In Situ Hybridization Method
		3.3 Immunofluorescence Staining
		3.4 Counterstain
		3.5 Reading Slides
	4 Attentions
	5 Discussion
	References
Chapter 2: Cell-Free RNA Sequencing from Biofluid Samples
	1 Introduction
	2 Materials
		2.1 Reagents
		2.2 Equipment
		2.3 Software
	3 Methods
		3.1 Cell-Free RNA Isolation
		3.2 1st Strand Synthesis
		3.3 2nd Strand Synthesis and Marking
		3.4 2nd Strand Synthesis and Marking Cleanup
		3.5 A-Tailing
			3.5.1 A-Tailing Immediately
			3.5.2 A-Tailing After Safe Stopping Point
		3.6 Adapter Ligation (See Note 6)
		3.7 1st Post-ligation Cleanup
		3.8 2nd Post-ligation Cleanup
		3.9 Library Amplification
		3.10 Library Amplification Cleanup
		3.11 Ribosome Depletion
		3.12 Pooling, Quantification, and Sequencing of Libraries
		3.13 Data Analysis
			3.13.1 LncRNA and mRNA Data Analysis
			3.13.2 miRNA Data Analysis
	4 Notes
	References
Chapter 3: Detection of Circulating Tumor DNA in Plasma Using Targeted Sequencing
	1 Introduction
	2 Materials
		2.1 Reagent
		2.2 Equipment
		2.3 Software
	3 Methods
		3.1 Sample Collection
		3.2 Prepare Cell-Free Plasma Samples
		3.3 Lysis with Proteinase K and cfDNA Isolation
		3.4 Wash with Wash Solution
		3.5 Wash with 80% Ethanol
		3.6 Elute the cfDNA
		3.7 cfDNA Quantification
		3.8 cfDNA End Preparation
		3.9 Adapter Ligation
		3.10 Purify the Sample with AMPure XP Beads
		3.11 Library Amplification
		3.12 Purify the Amplified Library with AMPure XP Beads
		3.13 Assess cfDNA Library Quality and Quantity
		3.14 Perform Hybridization Reaction
		3.15 Prepare Buffers
		3.16 Preparation and Activation of Streptavidin Beads
		3.17 Perform Bead Capture
		3.18 High-Temperature Washes
		3.19 Room-Temperature Washes
		3.20 Post-capture PCR
		3.21 Purification of the Post-capture Library with AMPure XP Beads
		3.22 Assess Post-capture Library Quality and Quantity
		3.23 NGS Sequencing
		3.24 Data Analysis
		3.25 Results of Reference Standard Samples
	4 Notes
	References
Chapter 4: Exosomal RNA Sequencing from Body Fluid Samples
	1 Introduction
	2 Materials
		2.1 Reagents
		2.2 Equipment
		2.3 Software
	3 Methods
		3.1 Plasma De-fibrination
		3.2 Exosome Isolation
		3.3 Assessment of Exosomes
		3.4 Extraction of Total Exosomal RNA
		3.5 Quality Control of Exosomal RNA
		3.6 Exosomal RNA Library Construction
			3.6.1 DNase Treatment and Primer Annealing
			3.6.2 First Strand cDNA Synthesis
			3.6.3 cDNA Processing
			3.6.4 Second Strand Synthesis
			3.6.5 End Repair
			3.6.6 Adapter Ligation
			3.6.7 Adapter Ligation Purification
			3.6.8 Library Amplification Optimization qPCR
			3.6.9 Library Amplification I
			3.6.10 Library Amplification I Purification
			3.6.11 Library Quantification
			3.6.12 rRNA Depletion
			3.6.13 Library Amplification II
			3.6.14 Library Amplification II Purification
		3.7 Quality Control of the Exosomal RNA Library
		3.8 Library Sequencing
		3.9 Data Analysis
			3.9.1 LncRNA Data Analysis
			3.9.2 miRNA Data Analysis
	4 Notes
	References
Chapter 5: Detection of Microorganisms in Body Fluid Samples
	1 Introduction
	2 Materials
		2.1 Reagents
		2.2 Equipment
	3 Methods
		3.1 Collection of Samples
		3.2 DNA Extraction
			3.2.1 DNA Extraction from Fecal Samples
			3.2.2 DNA Extraction from Body Fluid Samples
			3.2.3 Quality Control of Total gDNA
		3.3 16S Ribosomal RNA (rRNA) Gene Amplicons
		3.4 Shotgun Metagenomics
			3.4.1 DNA Shearing
			3.4.2 Library Construction: End Repair and A-Tailing
			3.4.3 Library Construction: Adapter Ligation
			3.4.4 Library Construction: Post-ligation Purification
			3.4.5 Library Construction: Library Amplification
			3.4.6 Library Construction: Post-amplification Cleanup
		3.5 Viral Integration Detection
			3.5.1 Library Construction
			3.5.2 Hybridization and Washing
			3.5.3 Prepare Bead Wash Buffer
			3.5.4 Prepare and Activate Streptavidin Beads
			3.5.5 Perform Bead Capture (See Note 7)
			3.5.6 High-Temperature Washes (See Note 8)
			3.5.7 Room-Temperature Washes
			3.5.8 Post-capture PCR
			3.5.9 Purify Post-capture PCR Fragments
			3.5.10 Quality Control of Library
		3.6 NGS Sequencing
		3.7 Bioinformatic Analysis
	4 Notes
	References
Chapter 6: The Interactome of Protein, DNA, and RNA
	1 Introduction
	2 Detection and Prediction of Protein-Protein Interactions (PPIs)
		2.1 Using Experimental Methods to Detect PPI
			2.1.1 Co-IP
			2.1.2 Pull-Down
			2.1.3 Genetically Encoded Residue-Selective Photo-Crosslinker
		2.2 Use some Software Tools to Detect and Predict PPI
			2.2.1 Proximity Labeling
			2.2.2 Network Analysis Software
			2.2.3 Machine Learning
		2.3 Database to Record PPI
			2.3.1 DIPs
			2.3.2 String
			2.3.3 BioGRID
	3 Experimental Methods, Software Tools, and Various Databases for Detecting and Predicting Interactions Between Proteins and D...
		3.1 Using Classical Experimental Methods to Detect Interactions Between Proteins and DNA
			3.1.1 ChIP
			3.1.2 EMSA
			3.1.3 Methylation Interference Assay
		3.2 Machine Learning-Generated Software Tools for Detecting and Predicting Protein-DNA Interactions
			3.2.1 Deepbind
			3.2.2 deepRAM
			3.2.3 FactorNet
			3.2.4 iDRBP_MMC
		3.3 The Interaction Relationship Between Protein and DNA Can be Obtained through some Databases
			3.3.1 Protein Data Bank (PDB)
			3.3.2 Uniprot
			3.3.3 3D Foot
	4 Use a Series of Methods to Capture and Record the Interaction Between Proteins and RNA
		4.1 Obtaining the Interaction Relationship Between Protein and RNA by Experimental Methods
			4.1.1 RNA Binding Protein Immunoprecipitation (RIP)
			4.1.2 RNA Pull-Down
		4.2 Use some Software Tools to Detect and Predict Protein-RNA Interactions
			4.2.1 Deepnet-Rbp
			4.2.2 iDeep
			4.2.3 EDCNN
		4.3 Some Databases Document the Interaction Between Protein and RNA
			4.3.1 EuRBPDB
			4.3.2 RBP2GO
			4.3.3 Circinteractome
	5 Summary
	References
Chapter 7: Liquid Biopsy in Bladder Cancer
	1 Introduction
	2 Circulating Tumor Cells (CTCs)
	3 Circulating Tumor DNA
	4 Urinary Tumor DNA
	5 Exosome
	6 Circulating RNA
	7 Conclusions
	References
Chapter 8: CSF Biopsy in Glioma: A Brief Review
	1 Introduction
	2 Overview of CSF Application in Glioma
	3 CTCs
	4 ctDNAs
	5 EVs
	6 miRNAs
	7 Conclusion and Prospects
	References
Chapter 9: Diagnosis, Monitoring, and Prognosis of Liquid Biopsy in Cancer Immunotherapy
	1 Introduction
	2 Utility of CIRCULATING FREE DNA (cfDNA) for Liquid Biopsy
	3 Utility of Circulating Tumor Cells (CTCs) for Liquid Biopsy
	4 Utility of EXOSOMES for Liquid Biopsy
	5 Utility of MicroRNAs (miRNAs) for Liquid Biopsy
	6 Discussion and Conclusion
	References
Chapter 10: The Implication of Liquid Biopsy in the Non-small Cell Lung Cancer: Potential and Expectation
	1 Background
	2 Definition and Introduction
	3 Supplementary Examination on Screening and Diagnosis Currently
	4 Comparison Between Traditional Tissue Biopsy and Liquid Biopsy
	5 Circulating Tumor Cell (CTC)
	6 Circulating Tumor DNA (ctDNA) and Cell-Free DNA (cdDNA)
	7 Extracellular Vesicles and Exosomes
	8 Conclusion and Expectation
	References
Chapter 11: Cell-Free DNA, MicroRNAs, Proteins, and Peptides as Liquid Biopsy Biomarkers in Prostate Cancer and Bladder Cancer
	1 Introduction
	2 Cell-Free DNA
		2.1 DNA Methylation
		2.2 Gene Mutation
	3 MicroRNA
	4 Proteins and Peptides
	5 Conclusion
	References
Chapter 12: Identification of Plasma Metabolites Associated with Lung Cancer Survival
	1 Introduction
	2 Materials
		2.1 Patients and Plasma Sample
		2.2 Solutions Used for Lipidomic Analysis
		2.3 Solutions Used for Metabolomic Analysis
		2.4 Materials for Reversed-Phase Liquid Chromatography-Mass Spectrometry (RPLC-MS) Analysis
		2.5 Materials for Hydrophilic Interaction Liquid Chromatography-Mass Spectrometry (HILIC-MS) Analysis
	3 Methods
		3.1 Study Design
		3.2 Metabolite Extraction for Lipidomic Analysis
		3.3 Metabolite Extraction for Metabolomic Analysis
		3.4 RPLC-MS Analysis
		3.5 HILIC-MS Analysis
		3.6 Metabolomics Data Analyses
		3.7 Statistical Analyses
	4 Results
		4.1 Patient Characteristics
		4.2 Plasma Metabolomics Analysis
		4.3 Prognostic Values of Metabolites
		4.4 Metabolites and Cancer Progression
	References
Chapter 13: The Detection of Exosomal PD-L1 in Peripheral Blood
	1 Introduction
	2 Exosomes as Tumor Biomarkers
		2.1 Exosomal Noncoding RNAs
		2.2 Exosomal mRNA
		2.3 Exosomal Proteins
	3 Detection of Exosome PD-L1 in Peripheral Blood
		3.1 ELISA
			3.1.1 Methods
		3.2 Droplet Digital PCR
			3.2.1 Methods
		3.3 Flow Cytometry
		3.4 SERS
		3.5 HOLMES-Exo PD-L1 Method
	4 Relationship Between Exosomal PD-L1 and Tumor Progression
	5 The Role of Exosomal PD-L1 in Tumor Immune Escape and Immunotherapy
	6 Conclusion
	References
Chapter 14: Liquid Biopsy in Hepatocellular Carcinoma
	1 Introduction
	2 CTCs in HCC
		2.1 CTCs Detection and Isolation
		2.2 CTCs as a Biomarker in HCC
	3 cfDNA in HCC
		3.1 Amount of cfDNA
		3.2 Mutations of cfDNA
		3.3 Methylation of cfDNA
	4 EVs in HCC
	5 Challenges and Prospects
	References
Chapter 15: Role of Circulating Tumor DNA in Colorectal Cancer
	1 Introduction
	2 Biological Characteristics of cfDNA and ctDNA
	3 Methodologies for Detection of ctDNA
	4 Clinical Applications of ctDNA
		4.1 ctDNA for Screening and Diagnosis
		4.2 ctDNA for Monitoring Prognostic and Recurrence
		4.3 ctDNA for Monitoring Drug Resistance
	5 Conclusion and Future Perspectives
	References
Chapter 16: Role of Liquid Biopsies in Rheumatoid Arthritis
	1 Introduction
	2 Blood-Based Liquid Biopsy and RA
		2.1 cfDNA in RA
			2.1.1 Recognition of cfDNA
			2.1.2 Genetic Variants and cfDNA
		2.2 Exosome in RA
			2.2.1 Serum Amyloid a (SAA)
			2.2.2 TLR-3
			2.2.3 miRNAs
			2.2.4 LncRNAs
	3 Other Types of Liquid Biopsy and RA
		3.1 Synovial Fluid
		3.2 Urine
		3.3 Saliva
	4 Future Perspectives and Conclusion
	References
Chapter 17: The Value of Cell-Free Circulating DNA Profiling in Patients with Skin Diseases
	1 Introduction
	2 Cell-Free DNA
	3 Melanoma
		3.1 BRAF
		3.2 Early Diagnosis
		3.3 Monitoring Disease Severity
		3.4 Monitoring Treatment Response
	4 Lymphoma
		4.1 B-Cell Lymphoma
		4.2 T-Cell Lymphoma
	5 Other Skin Malignancies
		5.1 Squamous Cell Carcinoma of Skin
		5.2 Extramammary Paget Disease
		5.3 Kaposi´s Sarcoma
	6 Non-Malignant Skin Diseases
		6.1 Psoriasis
		6.2 Systemic Lupus Erythematosus
		6.3 Pemphigus
	7 Conclusion
	References
Chapter 18: Circulating Non-coding RNAs and Exosomes: Liquid Biopsies for Monitoring Preeclampsia
	1 Introduction
	2 Origins of Placenta-Derived RNAs
	3 Definition and Functions of Non-coding RNAs
	4 Circulating ncRNAs in PE
		4.1 Potential of Circulating miRNAs as Early Markers of PE
		4.2 Potential of Circulating lncRNAs and CircRNAs as Early Makers of PE
	5 Circulating Exosomes as Potential Biomarkers of PE
		5.1 Biological Characteristics of Exosomes
		5.2 Circulating Placental Exosomal miRNAs in Normal Pregnancy
		5.3 Circulating Placental Exosomal miRNAs in PE
		5.4 Potential of Exosomal miRNAs in Peripheral Blood as Biomarkers for the Prediction of PE
	6 Conclusion and Perspectives
	References
Chapter 19: Liquid Biopsy in Coronary Heart Disease
	1 Introduction
	2 The Advantages of Liquid Biopsy
	3 Application of Liquid Biopsy in Individualized Diagnosis and Treatment of CHD
		3.1 Circulating Endothelial Cells and Endothelial Progenitor Cells
		3.2 Cell-Free DNA (cfDNA)
		3.3 Methylated DNA
		3.4 Circular RNAs (circRNAs)
		3.5 MicroRNAs (miRNAs)
		3.6 Extracellular Vesicles (EVs)
	4 Summary and Prospect
	References
Chapter 20: The Diagnostic and Prognostic Value of Synovial Fluid Analysis in Joint Diseases
	1 Introduction
		1.1 General Characteristics of Synovial Fluid Biopsy in Joint Diseases
		1.2 Synovial Fluid Biopsy in Infectious Joint Diseases
		1.3 Synovial Fluid Biopsy in Non-infectious Arthritis
		1.4 Synovial Fluid Biopsy in Traumatic Joint Diseases
		1.5 Synovial Fluid Biopsy in Other Joint Diseases
	2 Conclusions
	References
Chapter 21: Liquid Biopsy, a Potential New Detection Method in Heart Allograft Rejection
	1 Introduction
	2 Definition of Liquid Biopsy
	3 Liquid Biopsy and Heart Transplantation
		3.1 MHC Matching and Graft Rejection
		3.2 Cell-Free DNA (cfDNA) and Graft Rejection
		3.3 miRNA and Graft Rejection
		3.4 Other Biomarkers and Graft Rejection
	4 Conclusion
	References
Chapter 22: Potential Value and Application of Liquid Biopsy in Tumor, Neurodegeneration, and Muscle Degenerative Diseases
	1 Introduction
	2 The Development, Problems, and Application Progress of Liquid Biopsy in Tumor Field
	3 Current Status and Future Development Trend of Liquid Biopsy Research Outside Tumor Field
	4 Fluid Biopsy and Neurodegeneration
	5 AD
	6 PD
	7 Fluid Biopsy and Skeletal Muscle Degeneration
	8 Summary and Prospect
	References
Chapter 23: Liquid Biopsy in Adverse Neurodevelopment of Children: Problems and Prospects
	1 Introduction
	2 Fetal Period
		2.1 Brain Developmental Patterning and Common Congenital Neurological Defects Associated with Neurodevelopmental Disorders
		2.2 Liquid Biopsy in Fetal Period
			2.2.1 Cell-Free DNA (cf-DNA) and Cell-Free Fetal DNA (cff-DNA)
			2.2.2 DNA Methylation
			2.2.3 miRNA
			2.2.4 Metabolomics and Proteomics
	3 The Infant Period
		3.1 Brain Developmental Patterning and Common Injury in the Infant Period
		3.2 Screening and Diagnostic Methods for Neurodevelopment in Infancy
		3.3 Liquid Biopsy in Infant Period
	4 After Infant
		4.1 Screening and Diagnosis of Neurodevelopmental Disorders
		4.2 miRNA
	5 Conclusions and Future Perspectives
	References
Chapter 24: Advances of Liquid Biopsy for Diagnosis of Atrial Fibrillation and Its Recurrence After Ablation in Clinical Appli...
	1 Introduction
	2 Liquid Biopsy of AF and Its Recurrence
		2.1 SNP
			2.1.1 SNP and AF Occurrence
			2.1.2 SNP and AF Recurrence
		2.2 MicroRNAs (miRNAs)
			2.2.1 miRNAs and AF Occurrence
			2.2.2 miRNAs and AF Recurrence
		2.3 Mitochondrial DNA Copy Number (mtDNA-CN)
	3 Conclusions and Future Perspectives
	References
Index