Double-Stranded RNA: Methods and Protocols

Preface
Contents
Contributors
Chapter 1: Isolation of Double-Stranded RNAs by Lithium Chloride Fractionation
	1 Introduction
	2 Materials
		2.1 Chemical Reagents
		2.2 Extraction Buffer
		2.3 Equipment
	3 Methods
		3.1 Extraction of Total Nucleic Acids
		3.2 Extract Viral dsRNA by LiCl Fractionation
	4 Notes
	References
Chapter 2: Detection of dsRNA by Acridine Orange Staining
	1 Introduction
	2 Materials
		2.1 Plant Materials and Chemicals
		2.2 Equipment
		2.3 Buffer Solutions (See Note 1)
	3 Methods
		3.1 Preparation of Total  RNA
		3.2 DNase Treatment
		3.3 Gel Electrophoresis and Acridine Orange Staining
	4 Notes
	References
Chapter 3: Isolation of dsRNA from Plants by Cellulose Chromatography
	1 Introduction
	2 Materials
		2.1 Equipment
		2.2 Chemical and Regents
		2.3 Buffers
	3 Methods
	4 Notes
	References
Chapter 4: Rapid Purification of dsRNA Using Micro-Spin Cellulose Column
	1 Introduction
	2 Materials
		2.1 Equipment
		2.2 Chemical and Regents
		2.3 Buffers
	3 Methods
		3.1 Preparation of the Micro-spin Column Device
		3.2 Purification of dsRNA by Micro-spin Column
	4 Notes
	References
Chapter 5: Analysis of Virus-Induced Double-Stranded RNA in Living Plant Cells by the dRBFC Assay
	1 Introduction
	2 Materials
		2.1 Biological Materials
		2.2 Regents and Instruments
		2.3 Medium and Buffers
	3 Methods
		3.1 Preparation of N. benthamiana Seedlings
		3.2 Preparation of Agrobacteria
		3.3 Confocal Microscope Observation
		3.4 Results Analyses
	4 Notes
	References
Chapter 6: Detection of dsRNA with Fluorescence In Situ Hybridization (FISH)
	1 Introduction
	2 Materials
	3 Methods
		3.1 Viral Infection
		3.2 Protoplast Preparation
		3.3 Fixation
		3.4 Hybridization
		3.5 Observation
	4 Notes
	References
Chapter 7: Subcellular Colocalization Assay of Host Factors with Viral Replication Complex in the dsRNA Reporter Nicotiana ben...
	1 Introduction
	2 Materials
		2.1 Plasmids
		2.2 Plant Material and Bacterial Strains
		2.3 Equipment
		2.4 Reagents
	3 Methods
		3.1 Construction of the Red Fluorescent Protein-Tagged Host Gene via Ligation-Independent Cloning Method
		3.2 Introduction of Viruses and Host Genes into Plant Epidermal Cells Through Agroinfiltration
		3.3 Confocal Visualization of RFP-tagged Host Factor Localization with VRC in N. benthamiana B2:GFP
	4 Notes
	References
Chapter 8: Production of Double-Stranded RNA Using the Prokaryotic Promoter-Mediated Bidirectional Transcription
	1 Introduction
	2 Materials
		2.1 Primer Design and Amplification of Target Fragment
		2.2 Clone and Transformation
		2.3 Expression and Isolation of dsRNA
	3 Methods
		3.1 Clone of Target Genes
		3.2 Vector Construction
		3.3 Expression of dsRNA
		3.4 Isolation of dsRNA
	4 Notes
	References
Chapter 9: Inducible Expression of dsRNA in Escherichia coli
	1 Introduction
	2 Materials
		2.1 Equipment
		2.2 Chemical Reagents and Culture Medium
		2.3 Buffers
		2.4 Vector and Bacteria
	3 Methods
		3.1 Interference Fragment Cloning and Interference Vector Construction
		3.2 Induction of dsRNA Expression
		3.3 Extraction and Identification of dsRNA
	4 Notes
	References
Chapter 10: In Vivo Production of dsRNA Using Bacteriophage 훟6 in Pseudomonas syringae Cit7 Cells
	1 Introduction
	2 Materials
		2.1 Plasmids, Bacteria, and Plant Materials
		2.2 Equipment
		2.3 Chemical and Reagents
	3 Methods
		3.1 Plasmid Construction
		3.2 Electroporation
		3.3 In Vivo dsRNA Production
		3.4 Extraction of dsRNA
		3.5 Delivery of dsRNA into Plant Leaves
	4 Notes
	References
Chapter 11: Bacteria-Based Double-Stranded RNA Production to Develop Cost-Effective RNA Interference Application for Insect Pe...
	1 Introduction
	2 Materials
		2.1 Preparation of LB Liquid Medium
		2.2 Preparation of LB Solid Medium
		2.3 Preparation of Competent Cells
	3 Methods
		3.1 Obtaining the dsRNA Sequence Fragment of the Target Gene
		3.2 Construction of Recombinant Plasmids
			3.2.1 L4440 Plasmid Extraction
			3.2.2 Plasmid Ligation and Transformation
		3.3 dsRNA-Induced Expression
		3.4 dsRNA Extraction (see Notes 12 and 15).
			3.4.1 Phenol Extraction by Chloroform
			3.4.2 One-Step Extraction (75%Alcohol Precipitation Method)
			3.4.3 TRIzol Method
	4 Notes
	References
Chapter 12: Transiently Induce RNA Silencing in Plants Using a Tobacco Necrosis Virus A (TNV-A)-Based dsRNA Production System
	1 Introduction
	2 Materials
		2.1 Materials and Reagents
		2.2 Equipment
	3 Methods
		3.1 Plasmid Construction
		3.2 Plant Inoculation
		3.3 RNA Silencing Evaluation
		3.4 Results
	4 Notes
	References
Chapter 13: Co-immunoprecipitation-Based Isolation of Double-Stranded RNA-Associated Protein Complexes in Nicotiana benthamiana
	1 Introduction
	2 Materials
		2.1 Plant Material and Growth Conditions
		2.2 Preparation of Agrobacterium Culture
		2.3 Agrobacterium Infiltration Buffer
		2.4 Protein Extraction Buffer and Co-immunoprecipitation Reagents
		2.5 SDS-Polyacrylamide Gel Electrophoresis
		2.6 Western Blotting
	3 Methods
		3.1 Agrobacterium Infiltration
		3.2 Sample Preparation and Protein Extraction
		3.3 Co-Immunoprecipitation
		3.4 SDS-Polyacrylamide Gel Electrophoresis and Western Blotting Analysis
	4 Notes
	References
Chapter 14: Analysis of Plant Virus-Induced Immunity by Using Viral-Derived Double-Stranded RNA in Arabidopsis thaliana
	1 Introduction
	2 Materials
		2.1 In Vitro Synthesis of dsRNA
		2.2 Preparation of Arabidopsis thaliana Leaf Discs
		2.3 Protein Extraction and SDS-Polyacrylamide Gel Electrophoresis
		2.4 Western Blot
		2.5 Luminol-based Assay
	3 Methods
		3.1 In Vitro Synthesis of dsRNA
		3.2 Preparation of Arabidopsis thaliana Leaf Discs
		3.3 Poly(I:C) and dsRNA Treatment
		3.4 Protein Extraction and SDS-Polyacrylamide Gel Electrophoresis
		3.5 Western Blot
		3.6 Luminol-Based Assay
	4 Notes
	References
Chapter 15: Identification of Mycoviruses by dsRNA Extraction
	1 Introduction
	2 Materials
		2.1 Chemical and Reagents
		2.2 Culture Medium
		2.3 Buffers
	3 Methods
		3.1 Extraction of Viral dsRNA
		3.2 Assembly of the Viral Genome
		3.3 Sequencing and Phylogenetic Analyses
	4 Notes
	References
Chapter 16: Production of Double-Stranded RNA in Planta by a Potato Mop-Top Virus (PMTV)-Based Vector for Inducing Gene Silenc...
	1 Introduction
	2 Materials
		2.1 Materials
		2.2 Reagents and Buffers
		2.3 Equipment and Software
	3 Methods
		3.1 Determine the Target Fragment and Design Primer
		3.2 Construct of pCB301-PMTVR3 Harboring Fragment of Target Gene
		3.3 Infection of N. benthamiana
		3.4 Phenotype Observation and Silencing Efficiency Evaluation
		3.5 Results
	4 Notes
	References
Chapter 17: Application of dsRNA for Fungi Disease Management Sclerotinia sclerotiorum and Botrytis cinerea
	1 Introduction
		1.1 Considerations for the dsRNA Design
		1.2 Considerations for dsRNA Application Method
	2 Materials
	3 Methods
		3.1 S. sclerotiorum and B. cinerea Culture
		3.2 Acquisition of dsRNA/Synthesis of dsRNA by In Vitro Transcription
			3.2.1 DNA Templates for dsRNA Synthesis
			3.2.2 In Vitro Transcription dsRNA
		3.3 Application of dsRNA in Plants to Manage Fungal Disease
	4 Notes
	References
Chapter 18: Application of dsRNA in the Pine Wood Nematode, Bursaphelenchus xylophilus
	1 Introduction
	2 Materials
		2.1 Nematode Strain and Culture Conditions
		2.2 Acquisition of Nematodes at Different Development Stages
	3 Methods
		3.1 Synthesis of dsRNA
		3.2 RNAi by Soaking
		3.3 qPCR Detection
		3.4 Evaluate the Body Length of Adults After RNAi
		3.5 Representative Results
			3.5.1 Analysis of ppm-1 Expression of B. xylophilus After RNAi
			3.5.2 Effect of ppm-1 Expression on Growth and Development of B. xylophilus
	4 Notes
	References
Index