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RNA Editing: Methods and Protocols Ernesto Picardi
University of Hertfordshire Hatfield, Hertfordshire, UK
For further volumes: http://www.springer.com/series/7651
For over 35 years, biological scientists have come to rely on the research protocols and methodologies in the critically acclaimed Methods in Molecular Biology series. The series was the first to introduce the step-by-step protocols approach that has become the standard in all biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by step fashion, opening with an introductory overview, a list of the materials and reagents needed to complete the experiment, and followed by a detailed procedure that is supported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice. These hallmark features were introduced by series editor Dr. John Walker and constitute the key ingredient in each and every volume of the Methods in Molecular Biology series. Tested and trusted, comprehensive and reliable, all protocols from the series are indexed in PubMed.
Double-StrandedRNA
Methods and Protocols
Edited by Xiaofei Cheng
College of Plant Protection/Key Laboratory of Germplasm Enhancement, Physiology and Ecology of Food Crops in Cold Region of Chinese Education Ministry, Northeast Agricultural University, Harbin, China
Guanwei Wu
Institute of Plant Virology, Ningbo University, Ningbo City, Zhejiang, China
Editors
Xiaofei Cheng
College of Plant Protection/Key Laboratory of Germplasm Enhancement
Physiology and Ecology of Food Crops in Cold Region of Chinese Education Ministry
Northeast Agricultural University Harbin, China
Guanwei Wu Institute of Plant Virology
Ningbo University Ningbo City, Zhejiang, China
ISSN 1064-3745
Methods in Molecular Biology
ISSN 1940-6029 (electronic)
ISBN 978-1-0716-3701-2ISBN 978-1-0716-3702-9 (eBook)
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Preface
Double-stranded RNA (dsRNA) consists of two strands of RNA that are complementary to each other, forming a double-helix structure similar to DNA. It is the genetic material of some RNA viruses and the replication mediate of all RNA viruses. As a result, the presence of dsRNA is recognized as the hallmark of viral infection, and nearly all organisms have the capability of perception of dsRNA and mounting a response. For instance, dsRNA is recognized by Dicer or Dicer-like enzymes to trigger RNA interference (RNAi) in eukaryotic cells. In recent years, dsRNA-based approaches have emerged as a powerful tool for functional genomics research and have shown promise in combating viral infections and controlling pest populations in agriculture. As a result, dsRNA has gained significant attention in fields including plant biology, molecular biology, virology, entomology, and agriculture.
This volume is aimed to at providing detailed protocols concerning dsRNA. The volume consists of 18 protocols including classical and cutting-edge methods covering dsRNA isolation, visualization, characterization, production, and application. We tried to cover as many methods as possible for authors to select from. For instance, lithium chloride fractionation, cellulose chromatography, and a micro-spin column approach were characterized for dsRNA isolation in this volume. Each protocol is described in a step-by-step fashion to easily repeat and improve the protocol. Moreover, many protocols, such as coimmunoprecipitation-based isolation of double-stranded RNA-associated protein complexes, identification of mycoviruses by dsRNA extraction, application of dsRNA for fungi disease management (Sclerotinia sclerotiorum and Botrytis cin), and production of doublestranded RNA in plants by plant viral vectors for gene silencing, can also be easily adapted for identification of viruses from other organisms, control of other pathogens, and fundamental research.
We are grateful to all the authors for sharing their valuable working protocols. We apologize to the authors whose relevant work has not been cited and to readers for any missing dsRNA-related methods. Finally, we hope this volume will be a good reference book for students and researchers who work on dsRNA.
Harbin, ChinaXiaofei Cheng Ningbo City, Zhejiang, ChinaGuanwei Wu
1 Isolation of Double-Stranded RNAs by Lithium Chloride Fractionation
Mengzhu Chai, Xinyue Ma, Shuaifa Chang, and Xiaofei Cheng
2 Detection of dsRNA by Acridine Orange Staining
Tingshuai Ma, Yu Zhao, and Xiaofei Cheng
3 Isolation of dsRNA from Plants by Cellulose Chromatography
Yameng Luan, Wenqian Fan, Xayvangye Korxeelor, and Xiaoyun Wu
4 Rapid Purification of dsRNA Using Micro-Spin Cellulose Column
Yuting Wang, Ziyi Wang, and Xiaoyun Wu
5 Analysis of Virus-Induced Double-Stranded RNA in Living Plant Cells by the dRBFC Assay
Ying Zhang, Xinyue Fan, and Xiaofei Cheng
6 Detection of dsRNA with Fluorescence In Situ Hybridization (FISH) .
Wenling Zhou, Juan Huang, Xianguang Yang, and Xiaoming Zhang
7 Subcellular Colocalization Assay of Host Factors with Viral Replication Complex in the dsRNA Reporter Nicotiana benthamiana
Xinxin Fang, Jianping Chen, Fei Yan, and Guanwei Wu
8 Production of Double-Stranded RNA Using the Prokaryotic Promoter-Mediated Bidirectional Transcription
Xue Jiang, Kekely Bruno Attiogbe, Yating Guo, and Xiaoyun Wu
9 Inducible Expression of dsRNA in Escherichia coli
Saiya Duan and Guangjun Wang
10 In Vivo Production of dsRNA Using Bacteriophage ϕ6in Pseudomonas syringae Cit7 Cells
Jinguang Yang
11 Bacteria-Based Double-Stranded RNA Production to Develop Cost-Effective RNA Interference Application for Insect Pest Management 73
Ruobing Guan, Xuexia Miao, and Haichao Li
12 Transiently Induce RNA Silencing in Plants Using a Tobacco Necrosis Virus A (TNV-A)-Based dsRNA Production System
Yuanming Zhang, Mengzhu Chai, Xiaofei Cheng, and Kai Xu
13 Co-immunoprecipitation-Based Isolation of Double-Stranded RNA-Associated Protein Complexes in Nicotiana benthamiana 91
Zhaoxing Jia, Jianping Chen, Fei Yan, and Guanwei Wu
14 Analysis of Plant Virus-Induced Immunity by Using Viral-Derived Double-Stranded RNA in Arabidopsis thaliana 99
Penghuan Rui, Jianping Chen, Fei Yan, and Guanwei Wu
15 Identification of Mycoviruses by dsRNA Extraction 111
Yanfei Wang, Clement Nzabanita, and Lihua Guo
16 Production of Double-Stranded RNA in Planta by a Potato Mop-Top Virus (PMTV)-Based Vector for Inducing Gene Silencing . . . . . . . . . . 119
Ye Liu, Yanlin Gao, Xiaofei Cheng, and Yanju Bai
17 Application of dsRNA for Fungi Disease Management
Sclerotinia sclerotiorum and Botrytis cinerea 127
Tao Tang, Yumeng Wang, and Dongdong Niu
18 Application of dsRNA in the Pine Wood Nematode, Bursaphelenchus xylophilus 133
Chunyu Wang and Kai Guo
Contributors
KEKELY BRUNO ATTIOGBE • College of Plant Protection, Northeast Agricultural University, Harbin, China
YANJU BAI • Potato Research Institute of Heilongjiang Academy of Agricultural Sciences, Harbin, Heilongjiang, China
MENGZHU CHAI • College of Plant Protection, Northeast Agricultural University, Harbin, Heilongjiang, China; Key Laboratory of Germplasm Enhancement, Physiology and Ecology of Food Crops in Cold Region of Chinese Education Ministry, Northeast Agricultural University, Harbin, Heilongjiang, China
SHUAIFA CHANG • College of Plant Protection, Northeast Agricultural University, Harbin, Heilongjiang, China
JIANPING CHEN • State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, Institute of Plant Virology, Ningbo University, Ningbo, China
XIAOFEI CHENG • College of Plant Protection, Northeast Agricultural University, Harbin, Heilongjiang, China; Key Laboratory of Germplasm Enhancement, Physiology and Ecology of Food Crops in Cold Region of Chinese Education Ministry, Northeast Agricultural University, Harbin, Heilongjiang, China
SAIYA DUAN • State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China; Scientific Observation and Experimental Station of Pests in Xilin Gol Rangeland, Ministry of Agriculture and Rural Affairs, Xilinhot, China
WENQIAN FAN • College of Plant Protection, Northeast Agricultural University, Harbin, Heilongjiang, China
XINYUE FAN
• College of Plant Protection, Northeast Agricultural University, Harbin, Heilongjiang, China
XINXIN FANG
• State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, Institute of Plant Virology, Ningbo University, Ningbo, China
YANLIN GAO • Potato Research Institute of Heilongjiang Academy of Agricultural Sciences, Harbin, Heilongjiang, China
RUOBING GUAN • Henan International Laboratory for Green Pest Control, Henan Engineering Laboratory of Pest Biological Control, College of Plant Protection, Henan Agricultural University, Zhengzhou, China
KAI GUO • State Key Laboratory of Subtropical Silviculture, College of Forestry and Biotechnology, Zhejiang A & F University, Hangzhou, China
LIHUA GUO • State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China
YATING GUO
• College of Plant Protection, Northeast Agricultural University, Harbin, China
JUAN HUANG
• State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
ZHAOXING JIA • State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, Institute of Plant Virology, Ningbo University, Ningbo, China
XUE JIANG • College of Plant Protection, Northeast Agricultural University, Harbin, China
XAYVANGYE KORXEELOR • College of Plant Protection, Northeast Agricultural University, Harbin, Heilongjiang, China
YE LIU • College of Plant Protection, Northeast Agricultural University, Harbin, Heilongjiang, China; Potato Research Institute of Heilongjiang Academy of Agricultural Sciences, Harbin, Heilongjiang, China
HAICHAO LI • Key Laboratory of Insect Developmental and Evolutionary Biology, CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China
YAMENG LUAN • College of Plant Protection, Northeast Agricultural University, Harbin, Heilongjiang, China
TINGSHUAI MA • College of Plant Protection, Northeast Agricultural University, Harbin, Heilongjiang, China
XINYUE MA • College of Plant Protection, Northeast Agricultural University, Harbin, Heilongjiang, China
XUEXIA MIAO • Key Laboratory of Insect Developmental and Evolutionary Biology, CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China
DONGDONG NIU • Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, Nanjing, China; Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Nanjing, China
CLEMENT NZABANITA • State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China
PENGHUAN RUI • State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, Institute of Plant Virology, Ningbo University, Ningbo, China
TAO TANG • Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, Nanjing, China; Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Nanjing, China
CHUNYU WANG • State Key Laboratory of Subtropical Silviculture, College of Forestry and Biotechnology, Zhejiang A & F University, Hangzhou, China
GUANGJUN WANG • State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China; Scientific Observation and Experimental Station of Pests in Xilin Gol Rangeland, Ministry of Agriculture and Rural Affairs, Xilinhot, China
YANFEI WANG • State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China
YUMENG WANG • Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, Nanjing, China; Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Nanjing, China
YUTING WANG • College of Plant Protection, Northeast Agricultural University, Harbin, Heilongjiang, China
ZIYI WANG • College of Plant Protection, Northeast Agricultural University, Harbin, Heilongjiang, China
GUANWEI WU • State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, Institute of Plant Virology, Ningbo University, Ningbo, China
XIAOYUN WU • College of Plant Protection, Northeast Agricultural University, Harbin, Heilongjiang, China
KAI XU • Jiangsu Key Laboratory for Microbes and Functional Genomics, Jiangsu Engineering and Technology Research Center for Microbiology, College of Life Sciences, Nanjing Normal University, Nanjing, China
FEI YAN • State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, Institute of Plant Virology, Ningbo University, Ningbo, China
JINGUANG YANG • Key Laboratory of Tobacco Pest Monitoring, Controlling & Integrated Management, Tobacco Research Institute of Chinese Academy of Agricultural Sciences, Qingdao, China
XIANGUANG YANG • State Key Laboratory Base of Cell Differentiation and Regulation, College of Life Science, Henan Normal University, Xinxiang, China
XIAOMING ZHANG • State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, China; CAS Center for Excellence in Biotic Interactions, University of Chinese Academy of Sciences, Beijing, China; HainanYazhou Bay Seed Lab, Sanya, China
YING ZHANG • College of Plant Protection, Northeast Agricultural University, Harbin, Heilongjiang, China
YUANMING ZHANG • Jiangsu Key Laboratory for Microbes and Functional Genomics, Jiangsu Engineering and Technology Research Center for Microbiology, College of Life Sciences, Nanjing Normal University, Nanjing, China
YU ZHAO • College of Plant Protection, Northeast Agricultural University, Harbin, Heilongjiang, China
WENLING ZHOU • State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, China; CAS Center for Excellence in Biotic Interactions, University of Chinese Academy of Sciences, Beijing, China
Chapter 1
Isolation of Double-Stranded RNAs by Lithium Chloride Fractionation
Mengzhu Chai, Xinyue Ma, Shuaifa Chang, and Xiaofei Cheng
Abstract
This procedure provides a comprehensive method for isolating double-stranded RNA (dsRNA) that relies on the different solubility of various nucleic acids in lithium chloride (LiC1). The approach offers several notable advantages including simplicity, avoidance of enzymatic treatments, and the ability to obtain relatively high yields of undegraded dsRNA over other conventional techniques. Moreover, it allows for the separation of different groups of cellular and viral nucleic acids from a single tissue sample. This method was further improved to increase the purity of dsRNA using plant tissues infected by RNA viruses.
Key words Double-stranded RNA, Lithium chloride, Fractionation, Centrifugation, Virus detection
1 Introduction
Extraction of double-stranded RNA (dsRNA) is a critical first step in virus detection. Lithium chloride (LiCl) precipitation is a wellestablished method for extracting and purifying RNA from varied tissues including plant cells [1]. It offers several advantages such as selectivity for RNA over DNA and proteins and the ability to denature RNases that could degrade the RNA. There has been a growing interest in improving the efficiency and purity of RNA extractions, especially in the plant virology research field [2–4]. This is because plant samples often contain high levels of secondary metabolites that can interfere with RNA quality. The LiCl fractionation procedure was firstly proposed by Diaz-Ruiz and Kaper in 1978 and was remained unchanged for decades [1]. Recently, Henderson demonstrated a method to enhance the LiCl fractionation procedure by incorporating an additional washing step with longer incubation times, which resulted in increased RNA yield and purity [5]. Ananda and colleagues further developed a modified LiCl protocol specifically for RNA extraction from plant tissues that are rich in polysaccharides and polyphenols [6]. These advancements in
the LiCl-mediated RNA extraction methodology not only contribute to increasing the quality of the RNA yield but also expand the range of plant tissues from which high-quality RNA can be extracted. This chapter provides a comprehensive LiCl fractionation procedure for extracting dsRNAs from plant tissue infected by several well-known plant RNA viruses. Nevertheless, this method may be also useful for extracting dsRNAs from bacteria and animal tissue for diagnosing bacterial and animal viruses as well.
2 Materials
2.1 Chemical Reagents
All following chemicals are analytical grade. The manufacturers of the chemicals listed below are for reference only, and similar products from other companies can achieve the same effect. Store all chemicals at room temperature (unless indicated otherwise).
Prepare the following solutions using ultrapure water and store all reagents at room temperature (unless indicated otherwise). Diligently follow all waste disposal regulations when disposing waste materials. All three buffers can be used to extract total nucleic acid and can be selected appropriately.
1. Nucleic acid extraction buffer A: 0.1 M glycine, 0.01 EDTA, 0.1 M NaCl, pH 9.5, 10% SDS, and 5% Na bentonite [1].
2. Nucleic acid extraction buffer B: 0.1 M EDTA, 0.1 M TrisHCl, pH 8.9, 5% SDS, 40 mg/L Na bentonite [7].
3. Nucleic acid extraction buffer C: 0.2 M Tris-HCl, 0.4 M KCl, 0.2 M sucrose, 35 mM MgCl2, 25 mM EDTA, pH 9.0 [8].
11. Gel electrophoresis system (e.g., Jordan Scientific JP-250).
3 Methods
3.1 Extraction of Total Nucleic Acids
1. Pre-chill the mortar and pestle with liquid nitrogen (see Note 1).
2. Homogenize fresh or frozen virus-infected leaf tissues (50–100 g) in liquid nitrogen with a chilled mortar with a pestle into fine powder (see Note 2).
3. Mix the leaf powder with prechilled nucleic acid extraction buffers at a ratio of 1 g per 1 mL buffer (mass/volume).
4. Transfer the homogenate to 250 mL centrifuge bottle after adding 200 mL of water-saturated phenol containing 0.1% 8-hydroxyquinoline.
5. Shake vigorously for 2 min and then centrifuge at 6500 rpm for 10 min at 4 °C.
6. Carefully transfer the upper aqueous phase to a new tube (see Note 3).
3.2 Extract Viral
dsRNA by LiCl Fractionation
7. Repeat steps 4–6 twice until no white interface substance is visible.
8. Add two volumes of cold 95% ethanol to the aqueous phase.
9. Mix gently and then incubate at -20 °C for 2 h.
10. Centrifugation at 6500 rpm for 10 min at 4 °C.
11. Discard the supernatant (see Note 4).
12. Dissolve the pellet in distilled water (total nucleic acid solution).
1. Add 8 M LiCl to the total nucleic acid solution to the final concentration of 2 M (see Note 5).
2. Incubate on ice for 8–18 h and then centrifuge at 10,000 rpm for 15 min at 4 °C.
3. Transfer the supernatant to a new test tube and add about 1/2 volume of 8 M LiCl to a final concentration of 4 M LiCl (see Note 6).
4. Incubate on ice for 8–18 h and then centrifuge at 10,000 rpm for 15 min at 4 °C.
5. Discard the supernatant and resuspend the pellets in ~50 μL distilled water (see Note 7).
6. Analyze the dsRNA by gel electrophoresis, as in Fig. 1, and/or microvolume spectrophotometer (see Note 8).
7. Store the dsRNA fraction at -80 °C for future use.
Fig. 1 dsRNA profiles in 1% Agar gel. M, nucleic acid marker; Lane 1 and 2 showing the dsRNA extracted from two field samples showing viral-like symptoms by lithium chloride fractionation
4 Notes
1. Always wear disposable gloves and change frequently during the operation to prevent the contamination of your samples by bacteria, fungi, and RNase present on hands.
2. Make sure the leaf tissue is homogenized into fine powder for maximum dsRNA yield.
3. Make sure to get rid of all the white interface substance and the bottom phenol phase to avoid the contamination of proteins and acquire maximum dsRNA yield.
4. Make sure the pellet is retained.
5. LiCl precipitation requires high-quality RNA since the contaminations in low-quality RNAs may lead to degradation and even complete loss of dsRNA during the experiment; SDS, urea, and diatomaceous earth have certain inhibitory effects on RNases and can be used interchangeably with each other during the precipitation.
6. The pellet contains high-molecular-weight single-stranded RNAs (ssRNAs), which can be collected if needed.
7. The supernatant contains DNA and soluble low-molecularweight ssRNAs, which can be collected if needed.
8. Other nucleic acid analysis tools can also be used to estimate the quality of dsRNAs.
References
1. Diaz-Ruiz JR, Kaper JM (1978) Isolation of viral double-stranded RNAs using a LiCl fractionation procedure. Prep Biochem 8(1):1–17
2. Lot H, Kaper JM (1976) Physical and chemical dif ferentiation of three strains of cucumber mosaic virus and peanut stunt virus. Virology 74(1):209–222
3. Kaper JM, Siberg RA (1969) Degradation of turnip yellow mosaic virus by freezing and thawing in vitro: a new method for studies on the internal organization of the viral components and for isolating native RNA. Virology 38(3): 407–413
4. Morris TJ, Dodds JA (1979) Isolation and analysis of double-stranded RNA from virus-infected plant and fungal tissue. Phytopathology 69(8): 854–858
Henderson JM, Ujita A, Hill E et al (2021) Cap 1 messengerRNAsynthesiswith
co-transcriptional cleancap® analog by in vitro transcription. Curr Protocols 1(2):e39
6. Pratama AN, Grandmottet F, Rod-In W et al (2021) Efficient mini-prep RNA extraction from Dendrobium floral tissues rich in polysaccharides for validation of reference genes during flower development. Agric Natural Resour 55(3):349–358
7. Semancik JS, Weathers LG (1972) Exocortis vir us: an infectious free-nucleic acid plant virus with unusual properties. Virology 47(2): 456–466
8. Deng F, Xu R, Boland GJ (2003) Hypovirulence-associateddouble-stranded RNA from Sclerotinia homoeocarpa is conspecific with Ophiostoma novo-ulmi mitovirus 3a-Ld. Phytopathology 93(11):1407–1414
Detection of dsRNA by Acridine Orange Staining
Tingshuai Ma, Yu Zhao, and Xiaofei Cheng
Abstract
Acridine orange is a nucleic acid binding dye that emits green fluorescence when bound to double-stranded DNA or RNA and red fluorescence when bound to single-stranded DNA or RNA under ultraviolet light. This unique characterization allows it to be used for distinguishing or visualization of dsRNA. Here, we present a convenient and efficient protocol for detecting dsRNA in polyacrylamide gels.
Key words dsRNA, Acridine orange, In-gel staining
1 Introduction
Acridine orange (AO) is a fluorescent, nucleic acid intercalating dye that has been widely used in cytochemistry to track pH gradients in biological samples, nuclear fragmentations, flow cytometry, and other applications [1, 2]. AO was originally extracted from coal tar and was used as a dye and antibacterial agent in the fabric industry in the late nineteenth century [3, 4]. In 1940s, AO was discovered to have the capacity to bind nucleic acids [5]. Shortly, it was reported that AO has different affinities for double-stranded and single-stranded nucleic acids and emits different fluorescent signal after binding double-stranded and single-stranded acids: a green fluorescence at 530 nm when binds to double-stranded nucleic acids and red fluorescence at 640 nm when binds to single-stranded nucleic acids [6]. This unique property makes it an ideal agent for distinguishing double-stranded and singlestranded nucleic acids and stain cells [7, 8].
Double-stranded RNA (dsRNA) is the genetic material of dsRNA viruses and is produced during the replication of all RNA viruses. As a result, the presence of long dsRNA is recognized as a hallmark of virus infection. DsRNA is also crucial for numerous
biological functions of host cells such as triggering RNA silencing, signaling transporting, and processing of endogenous RNAs, e.g., microRNAs, transfer RNAs, and ribosome RNAs [9, 10]. AO emits red and green fluorescence when bound to single-stranded and double-stranded nucleic acids, respectively; while other nucleic acid dyes such as ethidium bromide (EB) lack color bias [11, 12]. This unique characteristic can be used to identify dsRNA from total RNAs and search for dsRNA from virus-infected cells [13, 14]. In this protocol, we provide a quick and reliable method for identifying dsRNA from total RNAs extracted from plant samples by AO staining in polyacrylamide gel.
7. Electrophoresis device (including casting tray and well combs).
8. UV transilluminator.
9. Microwave.
10. Flask.
11. Water bath or thermal cycle.
2.3 Buffer Solutions
(See Note 1)
1. CTAB buffer: 2.0% CTAB, 2.0% PVP 40, 1.4 M NaCl, 100 mM Tris-HCl (pH 8.0), 20 mM EDTA, and 1.0% betamercaptoethanol in RNase-free water.
2. CIA solution: mix 96 mL anhydrous chloroform and 4 mL isoamyl alcohol in biosafety hood at room temperature.
3. 8 M LiCl.
4. 1.0 M Tris-HCl (pH 8.0).
5. 0.5 M EDTA solution: weight 186.1 g EDTA and 18 g NaOH; stir the solution vigorously using a magnetic stirrer until EDTA dissolved completely; top up the solution to 1 L with DEPCtreated distilled water (see Note 2).
6. TE buffer: Measure out 1 mL 1M Tris-Cl (pH 8.0) and 0.2 mL 0.5 M EDTA (pH 8.0) to a 100 mL Duran bottle; Top up the solution to 100 mL by distilled RNase-free water.
7. 70% ethanol: Measure out 70 mL anhydrous ethanol and top up to 100 mL by distilled RNase-free water.
8. 50×TAE buffer: Weight 242 g Tris-base, measure out 57.1 mL 100% acetic acid, and 100 mL 0.5 M EDTA (pH 8.0); top up to 1 L by DEPC-treated distilled water.
3 Methods
3.1 Preparation of Total RNA
1. Preheat CTAB buffer to 65 °C before use.
2. Ground fresh leaf tissue into fine powder by a prechilled mortar and pestle or via a tissuelyser (see Note 3).
3. Transfer leaf powder to a 2 mL centrifuge tube containing 900 μL preheated CTAB buffer.
4. Gentle vor tex for 10 s.
10TingshuaiMaetal.
5. Incubate at 65 °C for 20 min on a shaker.
6. Centrifuge at 11,000 rpm for 10 min at room temperature.
7. Carefully transfer the supernatant to a new 2 mL centrifuge tube and add equal volume of CIA solution.
8. Vortex for 10 s by hand and centrifuge at 12,000 rpm for 10 min at room temperature.
9. Repeat steps 7 and 8 once.
10. Add prechilled 8 M LiCl to the final concentration of 4.0 M.
11. Mix gently and incubate at 4 °C overnight to precipitate RNA.
12. Centrifuge at 13,000 rpm for 20 min at 4 °C.
13. Discard the supernatant and add 500 μL prechilled 70% ethanol.
14. Mix gently and centrifuge at 13,000 rpm for 1 min at 4 °C.
15. Repeat steps 13 and 14 once.
16. Discard the supernatant and add 500 μL prechilled 100% ethanol.
17. Mix gently and centrifuge at 13,000 rpm for 1 min at 4 °C.
18. Discard the supernatant and dissolve the RNA pellets in 100 μL TE buffer.
3.2 DNase Treatment
3.3 Gel
Electrophoresis and Acridine Orange Staining
1. Measure 1 μg total RNA, add 1 μl10×DNase reaction buffer, 1 μL DNase I and top up to 10 μL by DEPC-treated distilled water (see Note 4).
2. Mix well and incubate at 37 °C for 15 min (see Note 5).
3. Add 1 μL of 25 mM EDTA (pH 8.0) and mix well by pipetting.
4. Incubate at 65 °C for 15 min to heat inactivate the DNase I.
5. Place on ice for 1 min; this RNA solution can directly be used for subsequent gel electrophoresis and reverse transcription reaction.
1. Measure 1 g of agarose and add 100 mL 1×TAE in a microwavable flask.
2. Microwave for 1–3 min until the agarose is completely dissolved (see Note 6).
3. Let agarose solution cool down to about 50 °C(see Note 7).
4. Add acridine orange to a final concentration of approximately 0.2–0.5 μg/mL.
5. Pour the agarose into a gel tray with the well comb in place (see Note 8).
6. Cold down the gel at 4 °C for 10–15 min or at room temperature for 20–30 min.
7. Place the gel into the gel tank and fill the tank will 1 TAB until the gel is completely covered.
8. Add equal volume of 2×RNA loading dye to each sample, mix well, and load into gel well.
9. Carefully load an RNA molecular weight ladder into the first lane of the gel set the electrophoresis unit.
10. Set up the electrophoresis set and run the gel at 90 V until the bromophenol blue is two-thirds of the way to the bottom (see Note 9).
11. Visualize the gel on a UV transilluminator and then use Photoshop CS to eliminate the blue channel that depicts the UV lamp (see Note 10).
4 Notes
1. Prepare all buffers with DEPC-treated or RNase-free water.
2. EDTA hardly dissolves under acid condition; sodium hydroxide should be added gradually to increase the solubility.
3. Make sure to ground the tissue into fine powder to achieve maximum dsRNA yield.
4. The reaction can be scaled up based on the total amount of RNA.
5. DNase treatment is often incomplete at 25 °C. DNase I is more effective at 37 °C.
6. Pulses model and swirling the flask occasionally is preferred to avoid overboiling the solution.
7. About when you can hold the flask (takes about 5 min).
8. Pour slowly to avoid bubbles; use the well-comb pipette tip to eliminate bubbles or push them to the sides of the gel if necessary.
9. Usually takes approximately 60 min to finish the electrophoresis.
10. A chemiluminescent imaging system with an adjustable light spectrum is more desirable.
References
1. Palmgren MG (1991) Acridine orange as a probe for measuring pH gradients across membranes: mechanism and limitations. Anal Biochem 192(2):316–321
2. Hitoshi Y, Lorens J, Kitada SI, Fisher J, LaBarge M, Ring HZ, Francke U, Reed JC, Kinoshita S, Nolan GP (1998) Toso, a cell
surface, specific regulator of Fas-induced apoptosis in T cells. Immunity 8(4):461–471
4. Browning CH, Cohen JB, Gaunt R, Gulbransen R (1997) Relationships between antiseptic
action and chemical constitution with special reference to compounds of the pyridine, quinoline, acridine and phenazine series. Proc R Soc Lond Ser B 93(653):329–366
5. Von Bertalanffy L, Bickis I (1956) Identification of cytoplasmic basophilia (ribonucleic acid) by fluorescence microscopy. J Histochem Cytochem 4(5):481–493
6. McMaster GK, Carmichael GG (1977) Analysis of single-and double-stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange. Proc Natl Acad Sci 74(11):4835–4838
7. Lillie RD, Conn HJ, Stotz EH, Emmel VM (1977) H. J. Conn’s biological stains: a handbook on the nature and uses of the dyes employed in the biological laboratory, 9th edn. Williams and Wilkins, Baltimore
8. Plemel JR, Caprariello AV, Keough MB, Henry TJ, Tsutsui S, Chu TH, Schenk GJ, Klaver R, Yong VW, Stys PK (2017) Unique spectral signatures of the nucleic acid dye acridine orange can distinguish cell death by apoptosis and necroptosis. J Cell Biol 216(4):1163–1181
9. Jacobs BL, Langland JO (1996) When two strands are better than one: the mediators and modulators of the cellular responses to doublestranded RNA. Virology 219(2):339–349
10. Ablasser A, Bauernfeind F, Hartmann G, Latz E, Fitzgerald KA, Hornung V (2009) RIG-I-dependent sensing of poly(dA:dT) through the induction of an RNA polymerase III–transcribed RNA intermediate. Nat Immunol 10(10):1065–1072
11. Carmichael GG, McMaster GK (1980) The analysis of nucleic acids in gels using glyoxal and acridine orange. Methods Enzymol 65(1):380–391
12. Bruno JG, Sincock SA, Stopa PJ (1996) Highly selective acridine and ethidium staining of bacterial DNA and RNA. Biotech Histochem 71(3):130–136
13. Pichlmair A, Schulz O, Tan CP, Rehwinkel J, Kato H, Takeuchi O, Akira S, Way M, Schiavo G, Reis e Sousa C (2009) Activation of MDA5 requires higher-order RNA structures generated during virus infection. J Virol 83(20):10761–10769
14. Lauretti F, Lucas de Melo F, Benati FJ, de Mello Volotao E, Santos N, Linhares RE, Nozawa C (2003) Use of acridine orange staining for the detection of rotavirus RNA in polyacrylamide gels. J Virol Methods 114(1): 29–35
Isolation of dsRNA from Plants by Cellulose Chromatography
Yameng Luan, Wenqian Fan, Xayvangye Korxeelor, and Xiaoyun Wu
Abstract
As the constitutive molecules of double-stranded RNA (dsRNA) virus genomes and replicative intermediates of single-stranded RNA (ssRNA) viruses, the high-molecular-weight dsRNAs are commonly found in RNA virus-infected plants. Therefore, the dsRNA is recognized as a hallmark of RNA virus infection and the profile of dsRNA has been applied as an efficient tool for diagnoses or characterization of unreported RNA viruses. Cellulose chromatography is one of the most useful procedures for the isolation of viral dsRNAs from total nucleic acids. Here, we describe rapid cellulose-based methods for purification of dsRNAs from plant tissue.
Key words Double-stranded RNA, Isolation methods, Cellulose chromatography, Plant virus
1 Introduction
Varied serological or polymerase chain reaction (PCR)-based diagnosis methods such as enzyme-linked immunosorbent assay (ELISA), Western blot, dot blot, reverse transcription-PCR (RT-PCR), and recombinase polymerase amplification-lateral flow assays (RPA-LFA) have been developed for rapid and reliable detection of viral diseases [4]. These techniques require antigen or sequence information of target viruses; thus, are not suitable for the diagnosis/detection of unknown viruses. The majority of all currently known viruses, especially plant viruses, contain RNA genomes [5]. The high-molecular-weight double-stranded RNAs (dsRNA) are constitutive materials of dsRNA virus genomes and the replicative intermediates of all RNA viruses. Therefore, the presence of dsRNA has been recognized as a hallmark of RNA virus infection [3, 7, 8]. With stability and easy extraction, dsRNA profiling is an efficient tool for diagnoses or characterization of RNA viruses but not reliable for viruses with DNA genomes
[9]. Moreover, the extracted dsRNA can be used for further the identification of unknown RNA viruses through high-throughput sequencing.
Several methods have been developed for the extraction of dsRNA such as cellulose chromatographic separation, enzymatic degradation of ssRNA and DNA, and lithium chloride fractionation [1]. Cellulose column chromatography is one of the most useful procedures for isolating viral dsRNAs from total nucleic acids. Here, we describe a rapid dsRNA extraction method using CF-11 cellulose and free of phenol and chloroform.
6. Nucleic acid extraction (NAE) buffer: 50 mM Tris-HCl (pH 8.5), 50 mM EDTA, 3% SDS, 1% β-mercaptoethanol, 1% PVPP-40.
7. 10×dsRNA-binding (10×DSB) buffer: 0.1 M Tris-HCl (pH 8.0), 1 M NaCl, 0.01 M EDTA.
8. 1×DSB buffer (containing 20% Ethanol): 10 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM EDTA, 20% ethanol.
9. Elution buffer: 10 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM EDTA.
10. 3 M sodium acetate (pH 5.2).
3 Methods
This protocol is based on the previous description with some modifications [2, 6]. Compared to phenol/chloroform extraction of dsRNA, this method is free of phenol and chloroform and is highly efficient and rapid (less than 2 h to complete whole steps and only requires about 200 mg of fresh material). All reagents and buffers should be prepared with DEPC-treated distilled water or directly with distilled RNase-free water (see Note 3).
1. Precool the mortar and pestle in liquid nitrogen.
2. Grind about 200 mg virus-infected plant sample in liquid nitrogen into fine powder (see Note 4).
3. Transfer the tissue powder to a 2 mL centrifuge tube containing 600 μL NAE buffer and add anhydrous ethanol to the final concentration of 30% (see Note 5).
4. Thaw the tissue powder by vortexing immediately and then shake the mixture gently for at least 20 min at room temperature on a horizontal shaker (see Note 6).
5. Centrifuge at 10,000× g for 20 min at 4 °C.
6. Equilibrate 40 mg cellulose CF-11 in 600 μL1×DSB buffer in a centrifuge tube (see Note 7).
7. Collect cellulose CF-11 by centrifuge and discard the supernatant thoroughly.
8. Transfer the supernatant of step 5 to the centrifuge tube containing equilibrated cellulose CF-11 from step 7
9. Mix well by gently vortexing, shake the mixture gently for 30 min at room temperature on a horizontal shaker (see Note 8).
10. Centrifuge at 5000× g for 1 min at room temperature to collect the cellulose CF-11.
11. Remove the supernatant and add 1 mL of 1×DSB buffer.
12. Mix well with a pipette and then shake gently for 5 min on a horizontal shaker at room temperature.
13. Repeat steps 10–12 twice (see Note 9).
14. Remove the supernatant completely by pipetting.
15. Add 200 μL elution buffer to the dried pellet, vortex gently for 5 min on a horizontal shaker, and then centrifuge at 5000× g for 1 min at room temperature.
16. Transfer the supernatant to a new 1.5 mL centrifuge tube kept on ice.
17. Repeat step 15 once.
18. Centrifuge at 5000× g for 1 min at room temperature to collect the supernatant to the same tube of step 16
19. Centrifuge the tube containing dsRNA for 1 min at 5000× g at 4 °C and transfer the supernatant to a new 1.5 mL tube to eliminate any remaining cellulose.
20. Add 1/10 volume of 3 M sodium acetate (pH 5.2) and 0.8 volume of isopropanol to the supernatant, mix well, and incubate overnight at -20 °Cor1hat -80 °C.
21. Centrifuge at >14,000× g,4 °C for 30 min to collect the dsRNA pellet.
22. Wash the dsRNA pellet with 70% ethanol twice by centrifugation.
23. Discard the supernatant, air dry the pellet in a biosafe cabinet for 10 min and dissolve the dsRNA pellet in about 200 μL DEPC-treated water (see Note 10).
4 Notes
1. SDS precipitates at low temperatures. The solution should be prepared with heating and needs to be warmed to at least room temperature prior to use.
2. EDTA is difficult to dissolve at high pH; therefore, it is necessary to measure the pH of the solution when adding HCl to adjust the pH.
3. Glass pipettes should be used to measure DEPC since some plastic pipettes can be dissolved by DEPC. DEPC treatment must be handled in a fume hood for safety. DEPC should be inactivated by autoclave at 121 °C for at least 15 min.
4. Use young and symptomatic leaf tissue and make sure to ground the leaf tissue into fine powder to maximize the dsRNA yield.
5. Use RNase-free pipette tips with a filter to avoid contamination among samples.
6. Avoid foam during vortex.
7. Equilibration can increase the binding activity of cellulose CF-11.
8. The incubation time can be increased.
9. The washing steps can be increased until the supernatant becomes colorless to eliminate the contamination of cytochrome.
10. The dsRNA solution may contain DNA and single-stranded RNAs, which can be eliminated by DNase and RNase treatment.
References
1. Atsumi G, Sekine K-T, Kobayashi K (2015) A new method to isolate total dsRNA. In: Uyeda I, Masuta C (eds) Plant virology protocols: new approaches to detect viruses and host responses. Springer, New York, pp 27–37
2. Balijja A, Kvarnhedenb A, Turchetti T (2008) A non-phenol-chloroform extraction of doublestranded RNA from plant and fungal tissues. Journal of Virological Methods 152:32–37
3. Dodds JA, Morris TJ, Jordan RL (1984) Plant viral double-stranded RNA. Annual Review of Phytopathology 22:151–168
4. Kalimuthu K, Arivalagan J, Mohan M, Christyraj JRSS, Arockiaraj J, Muthusamy R, Ju H-J (2022) Point of care diagnosis of plant virus: current trends and prospects. Molecular and Cellular Probes 61:101779
5. King AM, Lefkowitz E, Adams MJ, Carstens EB (2011) Virus taxonomy: ninth report of the International Committee on Taxonomy of Viruses. Elsevier
6. Marais A, Faure C, Bergey B, Candresse T (2018) Viral double-stranded RNAs (dsRNAs) from plants: alternative nucleic acid substrates forhigh-throughputsequencing.In: Pantaleo V, Chiumenti M (eds) Viral metagenomics: methods and protocols, vol 1746. Springer, New York, pp 45–53
7. Morris TJ, Dodds JA, Hillman B, Jordan RL, Lommel SA, Tamaki SJ (1983) Viral specific dsRNA: diagnostic value for plant virus disease identification. Plant Molecular Biology Reporter 1:27–30
8. Valverde R, Nameth S, Jordan R (1990) Analysis of double-stranded RNA for plant virus diagnosis. Plant Disease 74:255–258
9. Wu Q, Ding S-W, Zhang Y, Zhu S (2015) Identification of viruses and viroids by nextgenerationsequencingandhomologydependent and homology-independent algorithms. Annual Review ofPhytopathology 53: 425–444
Rapid Purification of dsRNA Using Micro-Spin Cellulose Column
Yuting Wang, Ziyi Wang, and Xiaoyun Wu
Abstract
Double-stranded RNA (dsRNA) is the replication intermediates of all RNA viruses. Purification and analysis of the profile and sequence of dsRNA is vital in virus diagnoses and/or characterization. Cellulose is one of the common materials used for isolation of dsRNA. Cellulose specifically binds dsRNA fraction under 15% ethanol concentration, which allows to isolate dsRNA from total nucleic acid solution or cell lysate. Here, we describe a rapid and reliable method for purifying dsRNA using a home-made micro-spin cellulose column from the cell lysate of virus-infected plant tissue. This labor-saving and rapid method enables routinely high-throughput isolation and analysis of dsRNA in plant or fungi samples.
Key words Double-stranded RNA, Isolation methods, Cellulose chromatography, Plant virus
1 Introduction
Precise diagnosis of viruses mainly relies on serological or polymerase-based virus diagnosis methods that have been developed such as immunochromatographic strip, enzyme-linked immunosorbent assay (ELISA), Western blot, reverse transcription-polymerase chain reaction (RT-PCR), and recombinase polymerase amplification-lateral flow assays (RPA-LFA) [1]. However, these methods can only detect known viruses. Traditionally, diagnosis of unknown viruses mainly relies on electron microscope and the combination of those serological or polymerase-based methods and even the experience of the researcher. The recently developed next-generation high-throughput sequencing (NGS) allows to discover known and unknown viruses without bias. Although total cellular RNA can be used for constructing of sequencing library for NGS, the mast number of host rRNA, small RNA, and mRNA greatly reduced the sensitivity of this method. RNA is the genetic material of most plant, animal, fungal, and bacterial viruses [2]. The replication of all RNA viruses
must go through the dsRNA phase as all genetic information in RNA is copied through base pairing similar as the DNA counterpart [3–5]. Therefore, dsRNA is the ideal material for NGS analysis of known and unknown RNA viruses [6]. Cellulose is one of the most powerful materials for isolating dsRNAs as it specifically and reversibly binds to dsRNA [7–9]. Here, we report simple and reliable dsRNA extraction methods using a home-made microspin cellulose column modified from Okada et al. [10].
2 Materials
2.1 Equipment
1. Glass measuring cylinders.
2. Mortar and pestle or electronic homogenizer (e.g., Qiagen TissueLyser II).
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Title: Compulsory manumission or, An examination of the actual state of the West India question
Author: Alexander McDonnell
Release date: February 19, 2024 [eBook #72991]
Language: English
Original publication: London: John Murray, 1827
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COMPULSORY MANUMISSION.
T H E A C T U A L S TAT E
OF THE W E S T I N D I A Q U E S T I O N .
BY ALEXANDER M‘DONNELL, Esq.
LONDON:
JOHN MURRAY, ALBEMARLE-STREET. MDCCCXXVII.
LONDON: PRINTED BY WILLIAM CLOWES, Stamford-Street.
ADVERTISEMENT.
It is fearful odds against a writer, when, at each stage of his task, he is liable to encounter prejudice as an upholder of a condition of society so repugnant to the feelings of Englishmen as that of slavery.
Greatly must those odds be increased, if a disposition be shown by Government, hitherto believed impartial, to array the weight of its authority against him.
But it is hoped that prejudice will not preclude inquiry. In the following pages it will be found, that in setting forth the actual state of the West India Question, the real and permanent welfare of the slaves occupies a conspicuous place.
In regard to the display of power, let us conclude, that when a measure can be demonstrated as positively bad, such disapprobation will be manifested by the independent and disinterested members of the legislature, as must exercise a salutary control over the counsels of ministers.
Under this impression, the following pages are respectfully submitted to the consideration of the members of both Houses of Parliament.
COMPULSORY MANUMISSION.
C I.
WEST INDIA PARTY DISINGENUOUSLY TREATED.
The West India Question is gradually narrowing to a point. There seems now to be little difference of opinion in regard to all safe and practicable measures tending to ameliorate the condition of the slaves, though the time and manner of their adoption may be dependent upon local considerations.
The question of emancipation, or that measure commonly designated Compulsory Manumission, alone remains at issue. The paramount importance of this clause, and the alarm felt in every West India colony at the threat of government to enforce its adoption, has caused the proceedings of the colonial department to be closely scrutinized, and it has in a variety of publications been charged with precipitation.
A pamphlet has lately appeared in vindication, under the title of “Remarks on an Address to the Members of the New Parliament, on the Proceedings of the Colonial Department with respect to the West India Question.” It is avowedly “written by a Member of the late Parliament,” and bears internal evidence of being the production of a gentleman connected with the Colonial Office.
This pamphlet calls for a reply, for two reasons: First, because the writer indulges in recrimination, and brings accusations against the West India body, which, if passed unnoticed, might produce a very erroneous impression on the minds of the moderate and disinterested portion of the legislature.
Secondly, because the writer discusses the compulsory manumission clause, and acquaints us with the nature and strength of the reasoning employed by government to justify the adoption of that measure.
The general tone of the publication will create much surprise, and in one respect it will be of service, in making known the true relations and influence of the contending parties. It has been asserted by the anti-colonial advocates, and believed by a large portion of the community, that the West Indians possessed great influence with government, by means of which their cause was powerfully strengthened.
If the well-informed portion of the public could once have entertained this belief, their error must appear manifest on a perusal of the pamphlet in question. The writer expresses himself very unceremoniously towards those of the West India body, who were members of the last parliament; and his tone might lead one to conclude that he thinks them not worth conciliation. He seems to justify his asperity, by complaining that the colonial department is improperly singled out for attack in regard to those proceedings which the colonial interests do not approve. Now he must be aware that, constitutionally speaking, responsibility peculiarly attaches to that officer of the crown from whose department particular acts emanate. If important measures affecting the colonies are carried into effect, while there is reason to believe that the Secretary of State for that department is in possession of despatches, official reports, or other information showing their inexpediency, he will be chiefly looked to for the consequences; because it is conceived to be his immediate duty to give full explanation of the details, both to his colleagues and to parliament, and not to incur responsibility for measures he could not conscientiously approve. These are rather the sentiments of the British nation than of any individual party.
It cannot, therefore, be invidious to canvass freely the acts of that particular department. It is the obvious and the regular course where grievances are felt; and all our ideas of public principle warrant a belief, that when such grievances are fairly stated, every officer of the crown to whose department they referred, so far from feeling indignant at their exposition, would be anxious to extend his protection, in order to have them promptly redressed.
Thus viewing the case, our advocate of the colonial department cannot mistake the tendency or application of any of the comments
contained in this publication; and he will be aware that a fair spirit of argument alone influences an examination of his positions, and of the judgment evinced in the manner and tone with which he has maintained them.
This writer endeavours to defend government, by charging the West Indians with inconsistency. This mode of argument, so frequently resorted to in political warfare, in nine cases out of ten indicates a feeble cause. It can surely never be too late to correct a principle radically wrong.
But let us examine the charge.
It is contended, that compulsory manumission was clearly laid down in the proceedings of parliament in March, 1824; that it was heard by the West India members without opposition, which was an implied acquiescence; and that if they now turn round to oppose it, they must have been “the most ignorant, incautious, and imbecile body of men who ever were got together to represent an interest.”
It was well known to the writer of this sentence, that the West India members were unanimously opposed to compulsory manumission; and it may be added, that a charge, couched in such language as this, could not have been expected from such a quarter. It may be true, that the West India members did not appeal to the House so early or so often as the threatened injustice may have demanded. But is this difficult to account for? When West India members have come forward to state their case, have we not seen it retorted upon them in the widely-disseminated publications of the anti-colonial party, in terms of the utmost coarseness: “He is a slave-driver: what attention or confidence is due to the statement of such a character?”
The West India members are indeed in a dilemma: if they speak, they run the risk of being abused—if silent, they are to be held parties to all the acts of precipitation and folly which may take place in colonial government.
It would be unnecessary to touch upon this point, did not one remark pre-eminently suggest itself for grave consideration. Throughout this pamphlet we have the defending of parties, clashing
of interests, and such terms; just as if the “Saints” and the West Indians were to fight the battle betwixt them, and whichever proved the most cunning, or the most persevering, would carry their point. And is this the language an apologist of government thinks it necessary to maintain? Can he have forgotten the grounds on which the West Indians were induced to commit their cause to the care of government? It was to stop useless or violent discussion. It proceeded from the principle, that if one party declaimed about alleged oppression of the negroes, and the other about their property, the safety of the colonies was the immediate province of ministers; that, as public servants, it was their sacred duty to uphold all the possessions of the crown; and that, in watching over their welfare, they equally protected the property of the colonists.
Though this consideration influenced the conduct of many West India members, still it is very erroneous to assert, that no explicit opposition was made to compulsory manumission at the very outset. It is asserted that it passed without animadversion, “except in a speech of the present Lord Seaford (Mr. C. R. Ellis), made on the day on which Mr. Canning uttered his celebrated commentary on its enactments.” One would think that dissent could not well be more clearly avowed than from the lips of the Chairman of the West India body. In point of fact, sufficient opposition, consistently with the respect which was shown to government, was evinced, to prove that the measure of compulsory manumission was from first to last peculiarly condemned by the colonial interests. When Mr. Canning first made known the intentions of government on this point, Lord Seaford explicitly denied being a party to them. When Mr. Brougham brought forward his last motion on the same subject, Lord Seaford again gave his reasons for resisting the measure. To those reasons, not a syllable in refutation was offered by Mr. Canning.
Besides these declarations of the chairman, every West India petition presented either to the king, to parliament, or to the Colonial office, explicitly set forth similar sentiments. The agents for the colonies, the merchants and mortgagees, early felt alarm; and in a petition from these last, presented on the 26th April, 1826, to the Lords by Lord Redesdale, and to the Commons by Mr. Baring, it was
stated, “That until it shall be proved, that free negroes will work for hire, the process of compulsory emancipation cannot even be experimentally commenced, upon a West India estate, with justice to the various parties holding legal claims upon the property.”
Surely nothing could be more explicit than this, to convey the opinion of the parties most deeply interested in the question. But the hardy assertion of the writer we have quoted, calls for still further explanation. This writer must know, that in every interview or communication with the Colonial office, those of the West India body, to whose opinions most weight was likely to be attached, were loud and strenuous in their entreaties for forbearance. They stated, that the government had not given sufficient examination to the case; that they were ignorant of many important local circumstances that those ought all first to be carefully and fully investigated before a measure of vital importance was enforced; and that, in short, the government were already getting into difficulties, and if they proceeded further they would get more deeply involved, and might find it unpleasant, if not impossible, to extricate themselves.
To attempt, therefore, to criminate the West India party, if opposition be now manifested, is not consistent with that impartiality which we have a right to expect from a member of the legislature; still less does it indicate the manly candour presumed to influence the conduct of an officer of His Majesty’s government.
It is argued, again, that a general declaration of dissent was not sufficient. The Order in Council for Trinidad contained four clauses, showing how compulsory manumission was to be carried into execution. That Order was uniformly described as the model for the rest of the colonies; and it was the duty of the West India party, in their subsequent proceedings, to move for the rescission of those clauses, if they entertained objections to them as a model.
Two reasons may be assigned why the colonists deemed it unnecessary to express their disapproval of the Trinidad order First, the tenure on which property was held in the British colonies differed from that of the Spanish colony of Trinidad. Secondly, it was notorious, that various portions of that order had to be repeatedly
sent home for explanation and remodelling. Was there, then, fair reason to believe, that every clause contained in the original order would be enforced upon the other colonies, while it was yet doubtful if they would be finally confirmed even in the colony for which the order had been originally framed?
Time was afforded the government for reflection. It was presumed, that they would learn, by experience, the difficulty occasioned by legislating precipitately for distant settlements. This line of conduct was dictated by respect for the government. Had the West India body in England come forward in a bolder manner than they had done to resist the determination avowed by the Colonial Secretary of State, how would they have been met? They would have been told by the very same parties who are now wilfully misinterpreting their quiescence—“You are exciting the colonial assemblies to needless opposition—your violence will engender contumacy in its worst form —remain silent till you know what is their decision.”
Such reflections undoubtedly did actuate them; but now, when opposition to compulsory manumission is known to be unanimous throughout the colonies which possess legislatures, the West India body in England, since repeated warnings and admonitions have been vain, do but consistently follow them up by appealing in a determined manner to parliament, and to the British nation, against the threatened intentions of government to enforce its enactment.
All the apologists of the measure seem, in their very tone, to be aware of the great degree of responsibility to be incurred in carrying it into effect; and hence there is a laboured attempt to show, that it is strictly conformable with the first proceedings of parliament, and with the resolutions of 1823. The West Indians are told, “in addition to your ignorance and imbecility, there will be the charge of shameful inconsistency to bring against you, if you now oppose our measures. You concurred unanimously in Mr. Canning’s resolutions; compulsory manumission is distinctly contemplated by those resolutions; and can you now propose to retract the assent given to them by your former votes.”
The West India members had little right to expect that such an accusation would ever be brought against them. In every stage of the proceedings, the whisper was incessantly reiterated in the ear of His Majesty’s ministers—“Adhere honestly to your own resolutions— for the sake of justice we call upon you, not to court a vulgar and transient popularity at our expense!”
And why was it that the resolutions were thus implicitly relied on? Not from any opinion that declarations of separate branches of the legislature could affect the rights of individuals resting on the statutelaws of the realm, but, because the parliamentary resolution contained a principle of cautious and practical legislation, and authorised the belief that, at each stage of procedure, careful examination and scrutiny would precede the adoption of measures which could be alleged, by any party concerned, to infringe their rights or interests.
When, subsequently, statements were made in the House of Commons, that government was departing from this principle, in enforcing compulsory manumission, neither Mr. Canning, nor Mr. Wilmot Horton, thought proper openly to attempt an explicit refutation.
What is the commentary? Matters are now becoming more critical, and the Executive resort to the plea of acting only upon the declared will of the legislature in their justification. Let us give them every advantage. Let us discuss the propriety of compulsory manumission, as it agrees with the resolutions of parliament; and if we succeed in our endeavours, we shall command the more attention, from meeting our antagonists on the ground they have themselves selected.
