Urinary Biomarkers: Methods and Protocols (Methods in Molecular Biology, 2292)

✍ Scribed by Samanta Salvi (editor), Valentina Casadio (editor)

• Publisher: Humana
• Year: 2021
• Tongue: English
• Leaves: 214
• Category: Library

✦ Synopsis

This volume describes important methods, protocols, and techniques used for studying urinary biomarkers. Chapters detail different alterations use to studied different types of cancers and physiological conditions. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Authoritative and cutting-edge, Urinary Biomarkers: Methods and Protocols aims to be a useful practical guide to researches to help further their study in this field.

✦ Table of Contents

Preface
Contents
Contributors
Part I: Urinary Biomarkers in Cancer
Chapter 1: Urinary Biomarkers in Tumors: An Overview
1 Introduction
2 Nucleic Acids
2.1 Methylation
2.2 Urine Cell-Free DNA
2.3 RNA-Based Biomarkers: The Example of PCA3
2.4 miRNAs
3 Proteins
4 Exosomes
5 Conclusions
References
Chapter 2: Urinary Cell-Free DNA Integrity Analysis
1 Introduction
2 Materials
3 Methods
3.1 DNA Samples Preparation
3.2 Samples and Primer Preparation for Real-Time PCR
3.3 Real-Time PCR
3.3.1 Data Analysis and Interpretation
4 Notes
References
Chapter 3: Mutational Analysis in Urinary Cell-Free DNA: KRAS in Colorectal Cancer
1 Introduction
2 Materials
2.1 ucfDNA Extraction and Quality Assessment
2.2 Mutational Analysis from ucfDNA
3 Methods
3.1 ucfDNA Extraction and Quality Assessment
3.2 ucfDNA Amplification
3.3 Setting Up the Instrument and Real-Time PCR
3.4 Data Analysis and Result Interpretation
4 Notes
References
Chapter 4: Fluorescence In Situ Hybridization in Urine Samples (UroVysion Kit)
1 Introduction
2 Materials
2.1 Materials Provided
2.2 Materials Required But Not Provided
2.3 Preparation of the Solutions
3 Methods
3.1 Specimen Collection
3.2 Specimen Processing
3.2.1 Sample Processing
3.2.2 Slide Preparation
3.2.3 Slide Pretreatment
3.3 FISH Procedure: UroVysion Assay
3.3.1 Manual Assay (See Note 6)
Denaturation of DNA Specimen
Probe Preparation
Hybridization
3.3.2 Optional Automated (HYBrite or ThermoBrite) Codenaturation Assay
Probe Preparation and Application
Denaturation of DNA Specimen and Hybridization on the HYBrite System
Denaturation of DNA Specimen and Hybridization on the ThermoBrite System
Post-hybridization Washes (Manual and Automated Assays)
3.4 Analysis of the Urine Samples
4 Notes
References
Chapter 5: Analysis of Copy Number Variation in Urine: c-Myc Evaluation Using a Real-Time PCR Approach
1 Introduction
2 Materials
2.1 Urinary Supernatant Collection
2.2 UcfDNA Isolation
2.3 UcfDNA Quantification
2.4 Real-Time PCR
3 Methods
3.1 Urinary Supernatant Collection
3.2 UcfDNA Isolation
3.3 ucfDNA Quantification Using Qubit Fluorometer
3.4 Real-Time qPCR Approach
3.4.1 Data Analysis and Interpretation
4 Notes
References
Chapter 6: Urinary microRNA and mRNA in Tumors
1 Introduction
2 mRNAs
3 miRNAs
4 Urinary Nucleic Acids in Bladder Cancer
4.1 mRNAs in Bladder Cancer
4.2 miRNAs in Bladder Cancer
5 Urinary Nucleic Acids in Prostate Cancer
5.1 mRNAs in Prostate Cancer
5.2 miRNAs in Prostate Cancer
6 miRNAs in Renal Carcinoma
7 Urinary miRNAs: A Challenge in Nonurological Tumors
7.1 Urinary miRNAs in Gastric Cancer
7.2 Urinary miRNAs in Female Tumors
8 Conclusions
References
Chapter 7: Long Noncoding RNAs as Innovative Urinary Diagnostic Biomarkers
1 Introduction
2 Long Noncoding RNAs for Bladder Cancer Diagnosis
2.1 UCA1
2.2 H19
2.3 LncRNA-Based Biomarker Panels
3 Long Noncoding RNAs for Prostate Cancer Diagnosis
3.1 PCA3
3.2 MALAT1 and FR0348383
3.3 LincRNA-p21
4 Conclusions
References
Chapter 8: Urinary Nucleic Acid in Tumor: Bioinformatics Approaches
1 Introduction
2 Analysis of Small Variants from ctDNA
3 cfDNA Molecule Tagging
4 Copy-Number Variants from cfDNA
5 Analysis of Epigenetic Profile in cfDNA
6 Conclusions
References
Chapter 9: PCA3 in Prostate Cancer
1 Introduction
2 PCA3
3 PCA3 as a Biomarker
4 The FDA-Approved Progensa PCA3 Assay
5 PCA3 and Prostate Biopsy
6 Conclusions
References
Chapter 10: Urinary Exosomes in Prostate Cancer
1 Introduction
2 Materials
2.1 Urinary Supernatant Collection
2.2 EVs Isolation
2.3 NanoSight Check
2.4 MACSPlex Exosome Kits
3 Methods
3.1 Urinary Supernatant Collection
3.2 EVs Isolation
3.3 Nanosight Check
3.4 MACSPlex Approach
3.4.1 Data Analysis and Interpretation
4 Notes
References
Chapter 11: Urinary Biomarkers In Bladder Cancer
1 Introduction
2 Current FDA-Approved Urinary Biomarkers
2.1 Fluorescent In Situ Hybridization (FISH): UroVysion
2.1.1 Defining the Cutoff for a Positive FISH Result
2.2 Nuclear Matrix Protein 22
2.3 Bladder Tumor Antigen
2.4 ImmunoCyt/uCyt +
3 Commercially Available (Not FDA-Approved) Urinary Biomarkers for Bladder Cancer
3.1 Cxbladder
3.2 AssureMDx
3.3 XPert BC
3.4 Urinary Bladder Cancer Rapid Test
3.5 Telomerase
4 Conclusion
References
Chapter 12: Telomerase Activity Analysis In Urine Sediment for Bladder Cancer
1 Introduction
2 Materials
2.1 Protein Isolation
2.2 TRAP ASSAY with Real Time PCR
3 Method
3.1 Urine Collection
3.2 Proteins Isolation and Quantification
3.3 Protein Quantification
3.4 TRAP Assay with Real Time PCR
4 Notes
References
Chapter 13: Protocols for Preparation and Mass Spectrometry Analysis of Clinical Urine Samples to Identify Candidate Biomarker...
1 Introduction
2 Materials
2.1 Urine Sample Storage
2.2 Methanol/Chloroform Precipitation
2.3 Estimation of Protein Concentration
2.4 In-Solution Tryptic Digest
2.5 Stage-Tip Desalting
3 Methods
3.1 Urine Sample Handling
3.2 Methanol-Chloroform Precipitation
3.3 In-Solution Tryptic Digest
3.4 Desalting Tryptic Peptides with C18
3.5 LC-MS/MS Measurement
4 Data Analysis
5 Notes
References
Part II: Urinary Extracellular Vesicles
Chapter 14: Current Methods for the Isolation of Urinary Extracellular Vesicles
1 Introduction
2 Serial Ultracentrifugation
3 Ultrafiltration
4 Polymer-Based Precipitation Methods
5 Density Gradient
6 Size-Exclusion Chromatography
7 Immuno-Bead Precipitation
8 Conclusion
9 Protocols
9.1 Serial Ultracentrifugation
9.2 Ultrafiltration
9.3 Polymer-Based Precipitation
9.4 Optiprep Density Gradient Ultracentrifugation
9.5 Size Exclusion Chromatography
9.6 Immunobead Protocol
References
Chapter 15: Urinary Extracellular Vesicles: Ultracentrifugation Method
1 Introduction
2 Materials
2.1 Collection and Preparation of Urine Samples
2.2 Ultracentrifugation with Sucrose-D2O Cushion
2.3 PS Affinity Method
3 Methods
3.1 Collection and Preparation Steps for Urine Samples
3.2 Ultracentrifugation
3.3 Sucrose-D2O Cushion Ultracentrifugation
3.4 PS Affinity Method for Isolation of EVs from Urine
3.5 Immobilization of the Biotinylated Tim4 to Streptavidin Magnetic Beads
3.6 Affinity Reaction
3.7 Washing of the EV-Binding Beads
3.8 Elution of the EVs
4 Notes
References
Part III: Physical Activity and Urinary Markers
Chapter 16: Urinary Catecholamines as Markers in Overtraining Syndrome
1 Introduction
2 Blood Biochemical Markers: An Overview
2.1 Plasma Glutamine
2.2 Blood Lactate Profile
2.3 Plasma Hormones
3 Urinary Markers: The Catecholamines
3.1 Urinary Catecholamines in Overtraining Syndrome
4 Conclusions
References
Chapter 17: Urinary Markers and Chronic Effect of Physical Exercise
1 Introduction
2 Markers of Kidney Disease and Damage
3 Physical Exercise and Urinary Biomarkers
4 Conclusions
References
Part IV: Urinary Metabolic Markers
Chapter 18: Urinary Metabolic Biomarkers in Cancer Patients: An Overview
1 Introduction
2 Hepatopancreatic Tract Cancers
2.1 Hepatocellular Carcinoma
2.2 Pancreatic Ductal Adenocarcinoma
3 Urinary Tract Cancers
3.1 Renal Cancer
3.2 Prostate Cancer
3.3 Bladder Cancer
4 Future Directions
References
Index