Circulating MicroRNAs: Methods and Protocols (Methods in Molecular Biology, 1024)

✍ Scribed by Nobuyoshi Kosaka (editor)

• Publisher: Humana
• Year: 2013
• Tongue: English
• Leaves: 275
• Category: Library

✦ Synopsis

microRNAs (miRNAs) are small non-coding RNAs that regulate various biological phenomena, such as development and homeostasis. The dysregulation of miRNA leads to disease progression, particularly of cancer. In Circulating MicroRNAs: Methods and Protocols, expert researchers in the field detail recent advances in the isolation, purification and analysis of circulating miRNAs from a variety of sources for research. The book is divided into three main topics. The first section involves the study of secretory miRNAs in cell-cell communication, and the second, the study of circulating miRNAs in body fluids. The last describes the novel techniques used to study circulating miRNAs. 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 key tips on troubleshooting and avoiding known pitfalls.

Authoritative and practical, Circulating MicroRNAs: Methods and Protocols seeks to aid scientists in dealing with the recent advances of RNAi technology from the bench to the bedside.

✦ Table of Contents

Preface
Contents
Contributors
Chapter 1: Functional Analysis of Exosomal MicroRNA in Cell–Cell Communication Research
1 Introduction
2 Materials
2.1 Transfection of Cell Lines for Exosome Isolation
2.2 Exosome Isolation by Differential Centrifugation
2.3 Determination of the Total Protein Concentration of the Exosome
2.4 RNA Isolation
2.5 miRNA qPCR
2.6 Animal Experiment
3 Methods
3.1 Transfection of Cell Lines for the Exosome Isolation
3.2 Exosome Isolation by Ultracentrifugation
3.3 Intratumor Injection of Exosome
3.4 RNA Isolation from Mouse Serum
3.5 cDNA Synthesis
3.6 miRNA PCR
4 Notes
References
Chapter 2: Isolation of Extracellular Nanovesicle MicroRNA from Liver Cancer Cells in Culture
1 Introduction
2 Materials
2.1 Nanovesicle Isolation from Cell Culture Medium of HCC Cell Lines
2.2 RNA Extraction from Tumor Cell-Derived Nanovesicles
2.3 Quality of RNA Extraction
3 Methods
3.1 Preparation of Vesicle-Depleted Medium ( See Note 2)
3.2 Isolation of Nanovesicles from Tumor Cell Culture Medium
3.3 RNA Extraction from Tumor Cell-Derived Nanovesicles
4 Notes
References
Chapter 3: Methods of Analysis of Dendritic Cell-Derived Exosome-Shuttle MicroRNA and Its Horizontal Propagation Between De...
1 Introduction
2 Materials
2.1 Animals
2.2 Materials and Reagents
3 Methods
3.1 Generation of Culture Supernatants Containing BMDC-Derived Exosomes
3.2 Concentration of Exosomes from Culture Supernatants of BMDCs ( See Note 6)
3.3 Purification of Exosomes from Culture Supernatants of BMDCs ( See Note 6)
3.3.1 Purification of Exosomes on a 30 % Sucrose/D 2 O Gradient
3.3.2 Purification of Exosomes on a Continuous Sucrose Gradient (Alternative)
3.4 Isolation and Identification of BMDC-Derived Exosome-Shuttle miRNAs
3.4.1 Exosome-Shuttle RNA Extraction
3.4.2 Assessment of Exosome-Shuttle RNA Concentration and Purity
3.4.3 Analysis of Exosome-Shuttle miRNAs by Real-Time RT-PCR
3.5 Functional Analysis of BMDC-Derived Exosome-Shuttle miRNAs
4 Notes
References
Chapter 4: Analysis of MicroRNA and Protein Transfer by Exosomes During an Immune Synapse
1 Introduction
2 Materials
3 Methods
3.1 Analysis of the Exchange of miRNA and Protein Between Immune Cells by Isolated Exosomes
3.1.1 Isolation and Characterization of Exosomes
Preparation of Exosome-Free Bovine Serum
Exosome Production and Isolation
Exosome Analysis and Quantification
3.1.2 Analysis of Exosomal miRNA and Protein Uptake by Recipient Cells
Incubation of Cells with Isolated Exosomes
Protein Uptake Analysis
miRNA Uptake Analysis
3.2 Analysis of miRNA and Protein Exchange After Immune Synapse
4 Notes
References
Chapter 5: Analysis of Viral MicroRNA Exchange via Exosomes In Vitro and In Vivo
1 Introduction
2 Materials
2.1 Transfection of Epithelial Cells for Exosome Isolation
2.2 Exosome Isolation by Differential Centrifugation
2.3 Labeling of Exosomes with PKH67 Green Fluorescent Linker Dye
2.4 Exosome Transfer Using a 1.0  m m Transwell Coculture System
2.5 RNase A Treatment
2.6 Separation B Cell/Non-B Cell Fraction from PBMCs
2.7 RNA Isolation
2.8 cDNA Synthesis
2.9 miRNA PCR
3 Methods
3.1 Transfection of Epithelial Cells for Exosome Isolation
3.2 Exosome Isolation by Differential Centrifugation
3.3 Labeling of LCL-Released Exosomes with PKH67 Green Fluorescent Linker Dye
3.4 Exosome Transfer Using a 1.0  m m Transwell Coculture System
3.5 RNase A Treatment
3.6 Separation B Cell/Non-B Cell Fraction from PBMCs
3.7 RNA Isolation
3.8 cDNA Synthesis
3.9 miRNA PCR
4 Notes
References
Chapter 6: Measurement of Precursor miRNA in Exosomes from Human ESC-Derived Mesenchymal Stem Cells
1 Introduction
2 Material
2.1 Isolation and Quantification of RNAs from Exosome and MSCs
2.2 Assessment of Exosomal miRNAs
2.3 Quantitative Analysis of Mature and pre-miRNA by qRT-PCR
3 Methods
3.1 Isolation and Quantification of RNAs from Exosome and MSCs
3.1.1 Extraction of RNAs from Exosomes
3.1.2 Extraction of Total RNAs from MSCs
3.1.3 Quantification of RNA Concentration
3.1.4 Enriching for Small RNA from Total MSC RNA Using the mir Vana™ miRNA Isolation Kit
3.1.5 Assessing the Molecular Weight Distribution of RNAs by Glyoxal Agarose Gel
3.1.6 Assessing the Molecular Weight Distribution of RNA by Tris-Borate-EDTA (TBE)-Urea Gels
3.2 Assessment of Exosomal miRNAs
3.2.1 Demonstrating That MSC Exosome RNAs Are Encapsulated in Cholesterol-Rich, Phospholipid Vesicles
3.2.2 Assessing the Presence of pre-miRNAs in Exosome RNA by RNase III-Susceptibility of 60–100 nt RNA Species
3.2.3 Identifying miRNAs in MSC Exosomes by Microarray Analysis
3.3 Quantitative Analysis of Mature and pre-miRNA by qRT-PCR
3.3.1 Quantitative Analysis of Mature miRNAs in MSCs and MSC Exosomes by qRT-PCR
3.3.2 Quantitative Analysis of pre-miRNA by qRT-PCR
3.3.3 Data Process and Calculation of the Ratio of pre-miRNA/miRNAs
4 Notes
References
Chapter 7: Analysis of the Transfer of Circulating microRNA Between Cells Mediated by Gap Junction
1 Introduction
2 Materials
2.1 Breast Cancer Cell Culture ( See Note 1)
2.2 Mesenchymal Stem Cell Culture ( See Note 1)
2.3 Transfection of Reporter Plasmid and pre-miRs ( See Note 1)
2.4 Cell Lysis and Detection of Luciferase Activity
2.5 Determination of Total Protein Concentration in Cell Lysates
3 Methods
3.1 Culture of MSCs
3.2 Culture of BCCs
3.3 Transfection of Cells with Reporter Construct
3.4 Coculture of Transfected MSCs and BCCs
3.5 Whole Cell Lysate Extraction
3.6 Luciferase Assay
3.7 Protein Assay and Calculation of Normalized Luciferase
4 Notes
References
Chapter 8: Isolation of Circulating MicroRNA Associated with RNA-Binding Protein
1 Introduction
2 Materials
2.1 Collection and Processing of Blood Plasma and Cell Culture Media
2.2 Immuno- precipitation
2.3 Isolation of Total RNA (Including miRNA) from Anti-AGO IP Pellets, Blood Plasma or Cell Culture Media
2.4 qPCR for Detection of Individual miRNAs
2.5 miRNA Profiling
3 Methods
3.1 Collection of Human Blood and Plasma Preparation
3.2 Preparation of Cell Culture Conditioned Media
3.3 Preparation of Exosomes-Free Blood Plasma or Cell Culture Media
3.4 Immuno- precipitation
3.5 MicroRNA Isolation
3.6 Detection of miRNA by qRT-PCR
3.7 miRNA Profiling
4 Notes
References
Chapter 9: Identification and Analysis of Circulating Exosomal microRNA in Human Body Fluids
1 Introduction
2 Materials
2.1 Collection of the Body Fluid
2.2 Exosome Isolation by Differential Centrifugation
2.3 Preparation of Exosomes for Electron Microscopy
2.4 RNA Isolation
2.4a RNA Isolation with Trizol  ‚
2.4b RNA Isolation with miRCURY™ kit
2.5 Analysis of the Quality and Concentration of RNA by Using the Agilent RNA 6000 Pico Kit and a Bioanalyzer
3 Methods
3.1 Collection of the Body Fluid
3.2 Exosome Isolation by Differential Centrifugation
3.3 Preparation of Exosomes for Electron Microscopy
3.4 RNA Isolation
3.4a RNA Isolation with Trizol ®
3.4b RNA Isolation with miRCURY™ Kit
3.5 Analysis of the Quality and Concentration of RNA by Using the Agilent RNA 6000 Pico Kit and a Bioanalyzer
4 Notes
References
Chapter 10: Analyzing the Circulating MicroRNAs in Exosomes/Extracellular Vesicles from Serum or Plasma by qRT-PCR
1 Introduction
2 Materials
2.1 Blood Collection Components
2.2 Extracellular Vesicle Purification Components
2.3 Flow Cytometry Components and Solutions
2.4 RNA Extraction Components
2.5 cDNA and Quantitative Real-Time PCR (qRT-PCR) Profiling Reagents and Solutions
3 Methods
3.1 Blood Collection and Processing
3.2 Quantification of Total Extracellular Vesicles
3.3 Fluorescent Surface Antigen Staining of Extracellular Vesicles
3.4 Analysis of Nucleic Acid Content and Annexin V Expression on Extracellular Vesicles
3.5 RNA Extraction Directly from Plasma or Serum Extracellular Vesicles
3.6 QIAcube Procedure
3.7 Optional: Manual Extraction Protocol
3.8 Ordering, Arrangement and Plating TaqMan ® microRNA PCR Primers in 384-Well Plates
3.9 Reverse Transcription (RT) for microRNA Analysis
3.10 Optional: Pre-Amplification Protocol
3.11 Quantitative Real-Time PCR for microRNAs
4 Notes
References
Chapter 11: Direct Serum Assay for MicroRNA in Cancer Patients
1 Introduction
2 Materials
2.1 Serum Isolation
2.2 RNA Extraction
2.3 RNA Quantification
2.4 RT-qPCR
2.5 RT-qPCR-DS
3 Methods
3.1 Serum Isolation
3.2 RNA Extraction
3.3 RNA Quantification
3.4 RT-qPCR
3.5 RT-qPCR-DS
3.6 Correspondence Between RT-qPCR and RT-qPCR-DS
4 Notes
References
Chapter 12: Analysis of MicroRNA Niches: Techniques to Measure Extracellular MicroRNA and Intracellular MicroRNA In Situ
1 Introduction
2 Materials
2.1 Sample Collection and Handling
2.2 Concentrating miRNA from Cell Culture Media
2.3 miRNA Extraction
2.4 qRT PCR
2.5 In Situ miRNA Detection
2.5.1 Buffers and Solutions
2.5.2 Wash Solutions
2.5.3 Reagents
2.5.4 Equipment
3 Methods
3.1 miRNA Measurement by RT-PCR
3.1.1 Sample Collection and Preparation: c-miRNA from Serum or Plasma
3.1.2 Sample Collection and Preparation: miRNA in Cell Culture Supernatant
3.1.3 Concentrating miRNA in Cell Culture Media
3.1.4 RNA Extraction
3.1.5 Reverse Transcription-Quantitative Polymerase Chain Reaction (RT-QPCR)
3.2 In Situ Staining of miRNA in Formalin-Fixed Paraffin-Embedded Tissue
3.2.1 Tissue Harvest and Fixation
3.2.2 Deparaffinization
3.2.3 Proteinase K Treatment
3.2.4 Formaldehyde Tissue Fixation
3.2.5 Fixation of Tissues with EDC ( See Note 11)
3.2.6 Wash Steps After Fixation
3.2.7 Acetylation for Inactivation of Enzymes in Tissues
3.2.8 Pre-hybridization and Hybridization
3.2.9 Post-hybridization Washes ( See Note 12)
3.2.10 Inactivation of Endogenous Peroxidase Activity
3.2.11 Anti-FITC Horseradish Peroxidase-Conjugation
3.2.12 Tyramide Amplification
3.2.13 Alkaline Phosphatase Detection System
3.2.14 Mounting Slides for Microscopy
4 Notes
References
Chapter 13: Analysis of Circulating MicroRNA by Microarray in Liver Disease
1 Introduction
2 Materials
3 Methods
3.1 RNA Preparation
3.2 Quality and Quantity of RNA Sample
3.3 Dephosphory-lation
3.4 Denature
3.5 Ligation and Cy3 Labeling
3.6 Prepare Hybridization Samples
3.7 miRNA Microarray
3.8 Data Analysis
4 Notes
References
Chapter 14: Isolation of Circulating MicroRNA in Saliva
1 Introduction
2 Materials
2.1 Saliva Collection Setup
2.2 For Whole Unstimulated Saliva
2.3 For Parotid Saliva Collection
2.4 For Submandibular Saliva Collection
2.5 For Exosome Isolation
2.6 For RNA Extraction
3 Methods
3.1 Saliva Collection Preparation
3.2 Unstimulated Whole Saliva Collection
3.3 Unstimulated Parotid and Submandibular Saliva Collection
3.4 Stimulated Parotid and Submandibular Saliva Collection
3.5 Exosome Isolation
3.6 RNA Extraction for the TRIZOL Isolation
4 Notes
References
Chapter 15: Purification of RNA from Milk Whey
1 Introduction
2 Materials
2.1 RNA Isolation and Analysis
2.2 Quantification of miRNA by qPCR
2.3 Quantification of mRNA by qPCR
3 Methods
3.1 Whey Fraction Preparation by Centrifugation (See Note 1)
3.2 Exosome Isolation by Ultracentrifugation (See Note 5)
3.3 RNA Isolation and Analysis ( See Note 2)
3.4 miRNA Quantification ( See Notes 3 and 6)
3.5 mRNA Quantification ( See Notes 4 and 6)
4 Notes
References
Chapter 16: Circulating MicroRNAs in the Cerebrospinal Fluid of Patients with Brain Diseases
1 Introduction
1.1 Production and Circulation of CSF
1.2 CSF Function
1.3 miRNAs in the Brain
1.4 Circulating miRNAs in CSF
2 Materials
2.1 Collection of Human CSF
2.2 mirVana PARIS Kit (Ambion) Including the Following Components
3 Methods (Fig.  1)
3.1 Collection of Human CSF
3.2 Sample Preparations
3.3 Isolate miRNAs from Human CSF
3.4 Confirm the Recovery and Functionality of miRNAs from Human CSF
3.5 Amplify Purified miRNAs in CSF and Determine the Ct Value
3.6 Select a Panel of Putative Normalizers to Be Analyzed
4 Notes
References
Chapter 17: Methods of MicroRNA Quantification in Urinary Sediment
1 Introduction
2 Materials
3 Methods
3.1 Preparation of Urine Samples
3.2 Collecting Microvesicle
3.3 RNA Extraction
3.4 Reverse Transcription
3.5 Real-Time Quantitative Polymerase Chain Reaction (RT-QPCR)
3.6 Measuring miRNAs Outside Microvesicles
4 Notes
References
Chapter 18: Circulating MicroRNA for the Identification of Forensically Relevant Body Fluids
1 Introduction
2 Materials
2.1 General Supplies and Equipment
2.2 Organic RNA Extraction ( See Note 1)
2.3 RNA Quantitation
2.4 Reverse Transcription
2.5 Real-Time PCR Detection
3 Methods
3.1 Organic RNA Extraction
3.2 RNA Quantitation
3.3 Reverse Transcription
3.4 Real-Time PCR Detection
3.5 Data Analysis
4 Notes
References
Chapter 19: Identification of Prostate Cancer-Associated MicroRNAs in Circulation Using a Mouse Model of Disease
1 Introduction
2 Materials
2.1 Mouse Serum Collection
2.2(a) miRNA Extraction for Downstream Microarray Analysis
2.2(b) miRNA Extraction for Downstream qRT-PCR Analysis
2.3 miRNA Profiling by Microarray
2.4 miRNA Quantitation by qRT-PCR
3 Methods
3.1 Mouse Serum Collection
3.2(a) miRNA Extraction: For Downstream Microarray Analysis ( See Note 4)
3.2(b) miRNA Extraction: For Downstream qRT-PCR Analysis ( See Note 6)
3.3 miRNA Profiling by Microarray ( See Note 11)
3.4 miRNA Quantitation by qRT-PCR
4 Notes
References
Chapter 20: A Combination of Extraction Reagent and DNA Microarray That Allows for the Detection of Global MiRNA Profiles f...
1 Introduction
2 Equipments/Materials/Reagents
2.1 Materials
2.2 Equipments
2.3 Reagents
3 Methods
3.1 miRNA Extraction
3.2 RNA Purification
3.3 RNA QC
3.4 miRNA Microarray
3.5 miRNA Data Analysis
4 Notes
References
Chapter 21: Nanopore Single-Molecule Detection of Circulating MicroRNAs
1 Introduction
2 Materials
2.1 Reagents
2.2 Setup and Instrument
3 Methods
3.1 Nanopore Formation
3.2 MiRNA Identification and Quantification in the Nanopore
3.3 Detection of miRNAs in RNA Extraction from Plasma
4 Notes
References
Index