MicroRNA Detection and Target Identification: Methods and Protocols

Preface
Contents
Contributors
Chapter 1: Detection of miRNAs
1 Introduction
2 Detection Technologies
2.1 General Challenges in Detecting miRNAs
2.2 Conventional Technologies for the Detection of miRNAs
2.2.1 Northern Blot
2.2.2 In Situ Hybridization
2.2.3 Microarray
2.2.4 RT-qPCR
2.2.5 Next-Generation Sequencing
2.2.6 In Vivo Reporter Assay
2.3 Emerging Technologies for the Detection of miRNAs
2.3.1 Oligonucleotide-Templated Reaction
2.3.2 Enzyme-Free Oligonucleotide Signal Amplification
2.3.3 Enzyme-Based Signal Amplification
2.3.4 Nanostructured Material-Based Amplification and Detection
2.3.5 Electrochemical Detection
2.3.6 Capillary-Electrophoresis-Based Detection
2.4 Point-of-Care (POC) Detection of miRNAs
2.4.1 Lateral Flow Assay Devices
2.4.2 Microfluidic Devices
3 Conclusion
References
Chapter 2: MicroRNA Identification, Target Prediction, and Validation for Crop Improvement
1 Introduction
2 Materials
3 Methods
3.1 Identification of MiRNA and Their Targets
3.1.1 Computational Analysis of Sequence Data
3.1.2 Prediction of Potential MiRNAs
3.1.3 Prediction of MiRNA Targets
3.1.4 Prediction of SSRs Within Pre-MiRNA Sequences
3.2 Primer Designing for MiRNA SSR Markers
3.3 In Silico PCR Analysis
3.4 DNA Isolation
3.4.1 Preparation of DNA Extraction Buffer
3.4.2 Homogenization and Incubation
3.4.3 Solvent Extraction
3.4.4 DNA Precipitation and Resuspension
3.5 RNase Treatment
3.6 Quantity and Quality Estimation of Isolated DNA
3.7 Polymerase Chain Reaction (PCR) Amplification
3.8 Visualization of Amplified Products
3.8.1 Agarose Gel Electrophoresis
3.8.2 Polyacrylamide Gel Electrophoresis
3.9 DNA Polymorphism and Allele Scoring
3.10 Molecular Data Analysis
3.11 Expression Analysis of Cluster Bean MiRNAs Using qRT-PCR
3.11.1 Total RNA Extraction Containing MiRNA
3.11.2 cDNA Synthesis
3.11.3 qRT-PCR-Based Expression Analysis
4 Notes
References
Chapter 3: MicroRNA Detection with CRISPR/Cas
1 Introduction
1.1 Application of Isothermal Amplification in MiRNA Detection
1.2 CRISPR/Cas-Based Techniques for Nucleic Acid Detection
2 Materials
2.1 Probe Preparation and Isothermal Amplification Reaction
2.1.1 Oligonucleotide Probes
2.1.2 Probe Preparation
2.1.3 Isothermal Amplification
2.2 E. coli Expression and Purification of Cas-Based Reporter Proteins
2.2.1 Plasmids Encoding Cas-Based Reporter Proteins
2.2.2 Protein Expression in E. coli
2.2.3 Purification of His-Tagged Protein from E. coli Lysate
2.3 In Vitro Transcription of sgRNAs
2.3.1 sgRNA/crRNA Coding Plasmids
2.3.2 In Vitro Transcription and Product Purification
2.4 In Vitro Cas Binding and Signal Output Assays
3 Methods
3.1 Probe Preparation and Isothermal Amplification Reaction
3.1.1 Probe Preparation and Validation for TIRCA Probes
3.1.2 EXPAR Reaction for Probe Validation
3.2 E. coli Expression and Purification of Cas-Based Reporter Proteins
3.2.1 Protein Expression in E. coli
3.2.2 Purification of Cas-Based Reporter Proteins
3.3 In Vitro Transcription and Purification of sgRNAs/crRNAs
3.3.1 In Vitro Transcription of sgRNAs/crRNAs
3.3.2 Purification of sgRNAs/crRNAs
3.4 In Vitro Cas Binding and Signal Output Assays
3.4.1 In Vitro Cas9 Binding and HRP Activity Assay for TIRCA-dCas9-Based miRNA Detection System (See Note 20)
3.4.2 In Vitro Cas12a Binding and Trans-Cleavage Assay for EXPAR-Cas12a-Based MiRNA Detection System
4 Notes
References
Chapter 4: Detection of MicroRNAs by Northern Blot
1 Introduction
2 Materials
2.1 RNA Extraction
2.1.1 Additional Reagents for RNA Extraction from Tissues with High Levels of Polysaccharides and Polyphenols
2.2 Denaturing PAGE
2.3 RNA Transfer
2.3.1 Additional Reagents and equipment for Capillary RNA Transfer
2.4 Cross-Linking
2.4.1 Additional Reagents and equipment for Chemical Cross-Linking
2.5 Hybridisation
2.5.1 Radioactive Hybridisation
2.5.2 Non-radioactive Hybridisation
3 Methods
3.1 RNA Extraction
3.1.1 RNA Extraction with TRIzol
3.1.2 Total Nucleic Acid Isolation from Plant Tissues with High Levels of Polysaccharides and Polyphenols
3.2 RNA Quality Control
3.3 Separation of RNA Samples by Denaturing 15% PAGE
3.4 Transferring RNA (See Note 16)
3.4.1 Capillary Blotting (See Note 17)
3.4.2 Semidry Blotting
3.4.3 Wet Blotting
3.5 Cross-Linking
3.5.1 UV Cross-Linking (See Note 19)
3.5.2 Chemical Cross-Linking
3.6 Hybridisation with Radioactive Probe (To Be Done in a Radioactive Lab)
3.6.1 Prehybridisation
3.6.2 Probe Preparation
3.6.3 G25 Sephadex (Size Exclusion) Column Preparation (See Note 24)
3.6.4 Hybridisation
3.6.5 Wash and Exposure
3.6.6 Stripping
3.7 Hybridisation with Non-radioactive Probe
3.7.1 Prehybridisation
3.7.2 Probe Preparation
3.7.3 Hybridisation
3.7.4 Washing
3.7.5 Detection
4 Notes
References
Chapter 5: MicroRNA Detection at Femtomolar Concentrations with Isothermal Amplification and a Biological Nanopore
1 Introduction
2 Materials
2.1 Isothermal Amplification
2.2 Nanopore Sensing
3 Methods
3.1 Isothermal Amplification
3.2 Nanopore Sensing
4 Notes
References
Chapter 6: Detection of MicroRNA Expression Dynamics Using LNA/DNA Nanobiosensor
1 Introduction
2 Materials
2.1 Cell Culture Material
2.2 Transfection
2.3 Reverse Transcription and RT-PCR
2.4 Equipment List
3 Methods
3.1 Design of LNA/DNA Probe for MiRNA Detection
3.2 Preparation of LNA/DNA Probe for MiRNA Detection
3.3 LNA/DNA Probe Transfection
3.4 Reverse Transcriptionand RT-PCR
3.4.1 Cell Lysis
3.4.2 Reverse Transcription
3.4.3 Real-Time PCR
3.4.4 Data Analysis
3.5 Detection of Endogenously and Exogenously Expressed MiRNAs
3.6 Detection of MiRNA Expression Dynamics in Living Cells
3.7 Multiplex Detection
4 Notes
References
Chapter 7: Programmable Ultrasensitive Molecular Amplifier for Digital and Multiplex MicroRNA Quantification
1 Introduction
2 Materials
2.1 PUMA Reaction Mixture
2.2 Droplet Microfluidics
2.3 Incubation and Droplet Imaging
3 Methods
3.1 Microfluidic Mold
3.2 Microfluidic Device
3.3 DNA Template Design
3.4 Digital PUMA Procedure
3.5 Droplet Incubation and Imaging
3.6 Droplet Analysis
4 Notes
References
Chapter 8: Small RNA Profiling by Next-Generation Sequencing Using High-Definition Adapters
1 Introduction
2 Materials
2.1 RNA Isolation
2.2 Adenylation of 3′ HD Adapter
2.3 3′ HD Adapter Ligation
2.4 5′ HD Adapter Ligation
2.5 Removal of Excess 3′ HD Adapter
2.6 cDNA Synthesis
2.7 PCR Amplification of cDNA
2.8 Gel Extraction of PCR Product
3 Methods
3.1 RNA Isolation
3.2 sRNA Purification
3.3 Adenylation of the 3′ HD Adapter
3.4 3′ HD Adapter Ligation
3.5 Removal of Excess 3′ HD Adapter
3.6 5′ HD Adapter Ligation
3.7 cDNA Synthesis and Amplification
3.8 Gel Electrophoresis and Extraction of PCR Products
3.9 In Silico Removal of Adapter Sequences
4 Notes
References
Chapter 9: Quantification of MicroRNAs or Viral RNAs with Microelectrode Sensors Enabled by Electrochemical Signal Amplificati...
1 Introduction
2 Materials
2.1 Electrode Preparation Component
2.2 SAM Preparation Component
2.3 Electrochemical Measurement
3 Methods
3.1 Preparation of Gold Microelectrodes
3.2 Monolayer Assembly of Recognition Probes on Electrode Surfaces
3.2.1 Higher Surface Coverage of Recognition Probes
3.2.2 Varied Surface Coverage of Recognition Probes
3.3 Electrochemical Detection of MiR-122
3.4 Measurement with Mismatched MiRNA Sequences
3.5 Diluted Mouse Serum Measurement
4 Notes
References
Chapter 10: Discovery and Evaluation of Extracellular MicroRNA Biomarkers in Plasma, Ascites, and Urine
1 Introduction
2 Materials
3 Methods
3.1 Body Fluid Collection
3.2 Separation of Extracellular Fraction
3.3 Isolation of Total RNA
3.4 Quality and Quantity Control of Total RNA
3.5 Large-Scale Screening and Validation of Individual MiRNAs
3.5.1 Large-Scale Screening
3.5.2 Validation of Individual MiRNA Expression
3.5.3 Analyses of MiRNA Expression
4 Notes
References
Chapter 11: Determining miRNA Expression Patterns in Xenopus
1 Introduction
2 Materials
2.1 Collection of Xenopus Species Embryos
2.2 Fixation of Xenopus Species Embryos
2.3 Whole Mount In Situ Hybridization
3 Methods
3.1 Collection of Xenopus Laevis Eggs
3.2 Fertilization of Xenopus Laevis Eggs
3.3 Collection of Xenopus Tropicalis Eggs
3.4 Fertilization of Xenopus Tropicalis Eggs
3.5 Fixation of Xenopus Species Embryos
3.6 LNA-Wish
3.7 Pri-MiRNA WISH
4 Notes
References
Chapter 12: Overview of Computational and Experimental Methods to Identify Tissue-Specific MicroRNA Targets
1 Introduction
2 MicroRNA Regulatory Complexity
3 MicroRNA-Target Prediction
3.1 Tissue-Specific MiRNA-Target Prediction
3.2 Features and Alterations that Affect MicroRNA-Target Interactions
4 Experimentally Supported MiRNA Target Identification
4.1 CLIP-Seq Enhances Identification of MiRNA Targets
4.2 Machine Learning-Based Target Prediction
5 MicroRNA Target Validation
5.1 Experimental Methods to Validate MiRNA-Target Predictions
5.2 Functional Characterization of the MiRNA Targetome
5.3 Construction of Regulatory Networks
6 Resources of MiRNA-Target Interactions
7 Conclusion
References
Chapter 13: sRNAtoolbox: Dockerized Analysis of Small RNA Sequencing Data in Model and Non-model Species
1 Introduction
2 sRNAtoolbox Docker Setup
2.1 Docker Engine Setup
2.2 Launching sRNAtoolbox Docker
2.3 Shared Folders
3 Automated Small RNA Expression Profiling and Analysis
3.1 Preparation of the Local Database
3.2 Parallelized Download of SRA Samples: fastq_down
3.3 MiRNA Expression Profiling: metaBench
4 Analysis of Putative Contaminations and Biologically Meaningful Presence of Exogenous RNA Molecules
5 Analysis in Non-model Species
5.1 Create Reference Sequences
5.2 Profile the Expression in a Single Sample
5.3 Parallelized Expression Profiling for Non-model Species
6 Study-Level Analysis and Differential Expression
6.1 metaDE
6.2 Analyze Output of sRNAbench-Microbes with sRNAde
6.3 Differential Expression in Non-model Species
7 Conclusions and Future Perspectives
References
Chapter 14: MicroRNA-Target Identification: A Combinatorial In Silico Approach
1 Introduction
2 Five Common Features of Mirna Target Prediction Tools
2.1 Seed Match
2.2 Conservation
2.3 Target Site Abundance
2.4 Site Accessibility
2.5 Free Energy
3 Proposed Method
3.1 Step 1: Prediction Based on Seed Match, Conservation, and Number of Target Sites in Each Transcript
3.2 Step 2: Evaluating Transcript Binding Status, Expression Pattern, and Disease Association
3.3 Step 3: Including MiRNA Targets That Are Experimentally Validated
3.4 Step 4: Venn Analysis for Selecting Mutually Predicted Genes
3.5 Step 5: Site Accessibility and Free-Energy Measurement
4 Synthesis
5 Notes
6 Future Recommendations
References
Chapter 15: An Efficient CRISPR-Cas9 Method to Knock Out MiRNA Expression in Xenopus Tropicalis
1 Introduction
2 Materials
2.1 Embryo Acquisition
2.2 Embryo Fixation
2.3 Embryo Injection
2.4 sgRNA Generation
2.5 MiRNA Mimics
2.6 Genomic DNA Isolation
2.7 Subcloning of PCR Products
2.8 qRT-PCR
3 Methods
3.1 Xenopus Husbandry and Embryo Acquisition
3.2 CRISPR-Cas9
3.2.1 Design of sgRNA
3.2.2 Generation of sgRNAs and CRISPR Reagents
3.2.3 Embryo Injection
3.3 Genotyping and Sanger Sequencing
3.4 Q-RT-PCR Validation of MiRNA KO
3.5 miRNA Rescue Experiment
4 Notes
References
Chapter 16: Interrogation of Functional miRNA-Target Interactions by CRISPR/Cas9 Genome Engineering
1 Introduction
2 Materials
2.1 sgRNA and ssODN Template Design Resources
2.2 sgRNA Cloning and ssODN Synthesis Reagents
2.3 Cell Culture and Transfection Reagents
2.4 RNA and DNA Extraction Reagents
2.5 High-Resolution Melt Analysis (HRMA) Components
2.6 T7 Endonuclease 1 (T7E1) Assay Reagents
2.7 RT-qPCR
3 Methods
3.1 sgRNA Design: miR-CRISPR
3.2 ssODN Design for HDR-Mediated MRE Replacement
3.3 sgRNA Cloning (pX330 Destination Vector)
3.4 MRE Engineering in HEK-293 T Cells
3.4.1 Introduction
3.4.2 HDR-Mediated MRE Replacement
4 Notes
References
Index