MicroRNA Profiling: Methods and Protocols

✍ Scribed by Sweta Rani

• Publisher: Humana Press
• Year: 2022
• Tongue: English
• Leaves: 258
• Series: Methods in Molecular Biology, 2595
• Edition: 2
• Category: Library

✦ Synopsis

This second edition provides updated and comprehensive methods on miRNA biogenesis and their role in the development and progression of various human diseases. Chapters detail miRNA biogenesis, isolating RNA, extracellular vesicles (EVs), circulating miRNAs, analyzing miRNA and miRDeep-P2, protocols for total RNA isolation from cells, cell-derived products, isolation and characterization of exosomes, serum, plasma specimens, and software tools. Written in the 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 protocols, and notes on troubleshooting and avoiding known pitfalls.

Authoritative and cutting-edge, MicroRNA Profiling: Methods and Protocols, Second Edition aims to provide comprehensive and accessible methods to undergraduate, graduate, and established scientist.

✦ Table of Contents

Preface
Contents
Contributors
Chapter 1: miRNA Biogenesis and Regulation of Diseases: An Updated Overview
1 Introduction
1.1 MicroRNAs (miRNAs)
2 miRNA Biogenesis
2.1 Transcription of miRNA
2.2 Formation of Pre-miRNA
2.3 Maturation of Pre-miRNA in Cytoplasm
3 Regulation of Dicer
3.1 Maturation of miRNA in RISC
4 Extracellular Vesicles (EVs)
4.1 Role of EVs and miRNA in Diseases
4.2 Neurodegenerative Diseases
4.3 Cardiovascular Disease
4.4 Cancer
5 Extracellular Vesicles (EVs) as Delivery Vehicle for miRNAs
5.1 Engineering EVs with miRNAs
5.2 Delivery of miRNA Using EVs
References
Chapter 2: Exosomal MicroRNA Profiling
1 Introduction
2 Exosome Isolation
2.1 Ultracentrifugation
2.2 Exosome Purification via Immunoaffinity Capture
2.3 Polyethylene Glycol (PEG)-Based Precipitation
2.4 Size Exclusion Chromatography (SEC)
2.5 Microfluidics-Based Techniques
3 Exosomal miRNAs
4 Exosomal miRNA Profiling
4.1 Nanomaterials
4.2 Microfluidics-Based Techniques
4.3 Detection of Exo-miRNAs in Single Exosomes
5 Purification and Normalization of Exosomal miRNAs
6 Conclusions
References
Chapter 3: MicroRNA Expression Profiling Using Agilent One-Color Microarray
1 Introduction
2 Materials
2.1 Required Equipment
2.2 Required Reagents
2.3 Reagent Required for miRNA Isolation
3 Methods
3.1 Total RNA, Including miRNA, Isolation
3.2 Sample Preparation and Labeling
3.3 Dry the Sample
3.4 Hybridization
3.5 Microarray Wash
3.6 Scanning and Feature Extraction
3.6.1 Slide Scan
3.6.2 Extract Data Using Agilent Feature Extraction Software
3.7 Normalizing Agilent One-Color Microarray Data
4 Notes
References
Chapter 4: A Simple Method for Profiling and Analyzing MicroRNAs from Small Volume Samples Using a qPCR-Based Platform
1 Introduction
2 Materials
2.1 Chemicals
2.2 Equipment
3 Methods
3.1 Small-Scale RNA Extraction
3.2 Generating Poly-A Tails, Adaptor Ligation, and Retro-Transcriptase Reactions
3.3 Pre-amplification
3.4 OpenArray Profile
3.5 Analysis of Data
4 Notes
References
Chapter 5: Exosomal MicroRNAs: Comprehensive Methods from Exosome Isolation to miRNA Extraction and Purity Analysis
1 Introduction
2 Exosomes Isolation by Differential Ultracentrifugation (dUC)
3 Electron Microscopy (EM)
4 Exo-miRNAs Content Analysis
5 RNA Quality Control
6 Materials
6.1 Required Equipment to Collect Media Conditioned by Cells
6.2 Buffer Required for Protein Extraction
6.3 Solutions Required for SDS-PAGE
6.4 Required Equipment for Protein Transfer
6.5 Required Equipment for Electron Microscopy
6.6 Reagent Required for Exo-miRNA Isolation
6.7 Required Equipment for RNA Quality Control
7 Methods
7.1 Exosome Isolation from Conditioned Media
7.2 Protein Extraction
7.3 SDS-Page
7.4 Protein Transfer
7.5 Immunoblotting
7.6 Electron Microscopy
7.7 Exo-miRNAs Content Analysis
7.8 RNA Quality Control
7.8.1 Gel Preparation
7.8.2 Gel-Dye Mix Preparation
7.8.3 Gel-Dye Mix Loading
7.8.4 Small RNA Conditioning Solution and Marker Loading
7.8.5 Sample Loading
8 Notes
References
Chapter 6: Detection of MicroRNAs in Brain Slices Using In Situ Hybridization
1 Introduction
2 Materials
2.1 Equipment
2.2 General Solutions
2.3 Solutions for Probes Hybridization
2.4 Solutions for Antibody Incubation
2.5 Development Signal Solution
3 Methods
3.1 Preparation of Brain Tissue for In Situ
3.2 Preparation of Slices for Hybridization
3.3 Hybridization of the Probes
3.4 Incubation with Primary Antibody
3.5 Incubation with NBT/BCIP Solution
4 Notes
References
Chapter 7: MicroRNA Profiling of Cell Lines and Xenografts by Quantitative PCR
1 Introduction
2 Materials
2.1 Cell Culture
2.2 miRNAs and Transfection Reagents
2.3 RNA Extraction Kit
2.4 RT miRNA Kit
2.5 qPCR Kit
3 Methods
3.1 Cell Culture
3.2 MiRNA Transfection
3.3 Establishment of Tumor Xenografts and In Vivo miRNA Delivery
3.4 RNA Extraction
3.5 RT (cDNA)
3.6 qPCR
3.7 Example
3.7.1 qPCR and Results Analysis
4 Notes
References
Chapter 8: Assessment of Basic Biological Functions Exerted by miRNAs
1 Introduction
2 Materials
2.1 Cell Line
2.2 Growth Media
2.3 Transfection Reagents
2.4 Acid Phosphatase Reagents
2.5 Equipment
3 Methods
3.1 miRNA Forward Transfection (Fig. 1)
3.2 Acid Phosphatase Assay (see Note 1)
3.3 Example Result
4 Notes
References
Chapter 9: Serum MicroRNAs Profiling in Age-Related Macular Degeneration
1 Introduction
2 Materials
2.1 Total RNA Extraction from Serum Using miRNeasy Serum/Plasma Kit (Qiagen, UK)
2.2 miRCURY LNA miRNA QC PCR Panel
2.3 miRCURY LNA miRNA SYBR Green qRT-PCR
3 Methods
3.1 Human Serum Preparation
3.2 RNA Extraction
3.3 Quality of Extracted RNA
3.3.1 First-Strand cDNA Synthesis Using the miRCURY LNA RT Kit
3.3.2 miRCURY LNA miRNA QC PCR Procedure for Quantitative PCR
3.3.3 Interpretation of the miRCURY LNA miRNA QC PCR Panel Data
3.4 miRCURY LNA miRNA SYBR Green Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)
3.4.1 First-Strand cDNA Synthesis
3.4.2 Quantitative, Real-Time PCR Using miRCURY LNA miRNA Custom PCR Panels
4 Notes
References
Chapter 10: Exosomal MicroRNA Discovery in Age-Related Macular Degeneration
1 Introduction
2 Materials
2.1 Total RNA Extraction from Serum
2.2 The miRCURY LNA Universal RT MicroRNA PCR for Serum (Exiqon)
2.3 Cell Culture of Retinal Pigment Epithelium Cells
2.4 Exosomes Isolation from Serum
2.5 Validated Antibody Systems for Confirmation of Exosomes (Western Blot Analysis)
2.6 RNA Extraction from Cells
2.7 Cytotoxicity Assays
2.8 The Human Apoptosis miScript miRNA PCR Array (Qiagen)
2.8.1 Reverse Transcription for Quantitative Real-Time PCR (qRT-PCR)
2.8.2 Real-Time PCR for Mature miRNA Expression Profiling
2.9 Angiogenesis Assays
3 Methods
3.1 Human Serum Preparation
3.2 RNA Extraction
3.2.1 RNA Isolation and Quantification
3.2.2 Interpretation of Results
3.3 The miRCURY LNA Universal RT MicroRNA PCR for Serum Using the Exiqon Platform
3.3.1 First-Strand cDNA Synthesis
3.3.2 MicroRNA Real-Time qPCR Amplification
3.3.3 Data Quality Control
3.3.4 Identification of Consistent Normalizer (Endogenous Control) for Normalization
3.3.5 Normalization
3.3.6 Data Analysis
3.4 Bioinformatics
3.5 Functional Analysis
3.5.1 Experimental Design
3.5.2 Cell Culture of Retinal Pigment Epithelium Cells
3.5.3 Exosome Isolation from Serum Using ExoQuick
3.5.4 Track Exosomes with Validated Antibody Systems
3.6 ARPE-19 Exosome Treatment, Cell Pellet Collection, and RNA Extraction
3.6.1 GenElute Mammalian Total RNA Miniprep Protocol
3.7 Apoptosis Assay
3.7.1 Cytotoxicity Assays in ARPE-19 Cells Treated with AMD-Derived Exosomes
3.7.2 Human Apoptosis miScript miRNA PCR Array Profiling in ARPE-19 Cells Treated with AMD-Derived Exosomes
4 Data Analysis
4.1 Angiogenesis Assays on Human Endothelial Cells Treated with AMD-Derived Exosomes
5 Notes
References
Chapter 11: MicroRNA Profiling Using a PCR-Based Method
1 Introduction
2 Materials
2.1 Primer Dilution
2.2 RNA Extraction
2.3 DNAse Treatment
2.4 cDNA Synthesis
2.5 MiRNA RT-qPCR
3 Methods
3.1 Primers Design and Dilution
3.2 RNA Extraction
3.3 Eliminating Contaminant DNA after RNA Isolation
3.4 cDNA Synthesis and miRNA RT-qPCR
3.4.1 Poly(a) Tailing Reaction
3.4.2 First-Strand cDNA Synthesis Reaction
3.5 MiRNA RT-qPCR
3.6 Data Analysis
4 Notes
References
Chapter 12: Guidelines on Designing MicroRNA Sponges: From Construction to Stable Cell Line
1 Introduction
2 Materials
2.1 Construction of miRNA-30a Sponge
2.2 Cloning of SanDI: pEGFP-C3/Linker
2.3 Cloning of miR30a and Scrambled Sponges
3 Methods
3.1 Cloning of SanDI
3.2 Cloning of miR30a and Scrambled Sponges
3.3 Generation of Stable Genetic Models of miR-30a
4 Notes
References
Chapter 13: Evaluating Single-Nucleotide Variants in MicroRNA Targeting Sites and Mature MicroRNA In Vitro Cell Culture by Luc...
1 Introduction
2 Materials
2.1 Samples: DNA Isolation
2.2 Sample Preparation: Conventional PCR
2.3 Vectors
2.4 Clones´ Constructions: miR-146a + pcDNA3.3 and 3′-UTR-BRCA2 + pMIR-REPORT Luciferase
2.4.1 Inserts (miR-146a-Sequence and 3′-UTR-BRCA2) and Empty Vectors (pcDNA3.3 and pMIR-REPORT Luciferase Vectors): Purificati...
2.4.2 pcDNA3.3 + miR-146a And pMIR-REPORT +3′-UTR BRCA2: Ligation
2.4.3 Bacteria Transformation
2.5 Co-transfections into Cell Lines
3 Methods
3.1 Samples: Isolation of DNA
3.2 Sample Preparation: Conventional PCR
3.3 Clone Constructions: miR-146a + pcDNA3.3 and 3′-UTR-BRCA2 + pMIR-REPORT Luciferase
3.3.1 Inserts (miR-146a-Sequence and 3′-UTR-BRCA2) and Empty Vectors (pcDNA3.3 and pMIR-REPORT Luciferase Vectors): Purificati...
3.3.2 pcDNA3.3 + miR-146a And pMIR-REPORT +3′-UTR-BRCA2: Ligation
3.3.3 Bacteria Transformation
3.4 Luciferase
3.5 Luciferase Results
4 Notes
References
Chapter 14: Assessment of Cell Cytotoxicity in 3D Biomaterial Scaffolds Following miRNA Transfection
1 Introduction
2 Materials
2.1 Cell Line
2.2 Scaffold Fabrication
2.3 Growth Media
2.4 Transfection Reagents
2.5 Cell Viability
3 Methods
3.1 Scaffold Fabrication
3.2 Seeding Collagen-Based Scaffolds
3.3 Gene Activation of Collagen-Based Scaffolds
3.4 Cytotoxicity Assay
4 Notes
References
Chapter 15: Evaluation of miRNA Expression in 3D In Vitro Scaffold-Based Cancer Models
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Transfection Reagents
2.3 Gene Expression Analysis Reagents
2.4 PicoGreen Reagents
2.5 Equipment
3 Methods
3.1 Assembly of Cells on Scaffolds
3.2 miRNA Forward Transfection
3.3 RNA Extraction
3.4 Gene Expression Analysis
3.5 PicoGreen dsDNA Assay
3.6 Example Results
4 Notes
References
Chapter 16: Bioinformatics Analysis of miRNA Sequencing Data
1 Introduction
2 Materials
3 Methods
3.1 FASTQ
3.2 Quality Control of the FASTQ Files
3.3 Trimming Reads to Remove Adapters
3.4 miRNA Alignment and Quantification
3.5 miRDeep2
3.6 Generating Precursor Expression Matrix for All Samples
3.7 Differential Expression Analysis
References
Chapter 17: Plant MicroRNA Identification and Annotation Using Deep Sequencing Data
1 Introduction
2 Materials
2.1 sRNA-Seq Datasets
2.2 Installation and Computational Resources
3 Method
3.1 Overview of miRNA Prediction and Annotation
3.2 miRNA Prediction
3.2.1 Installation
3.2.2 Reads Cleaning
3.2.3 Genome Indexing
3.2.4 miRDP2 Prediction
3.2.5 Detailed Processes of miRDP2
3.3 miRNA Annotation
3.3.1 miRDP2 Annotation
3.3.2 Detailed Processes in the Pipeline
4 Notes
References
Index