Molecular Techniques for Studying Viruses: Practical Notes

Preface
Acknowledgements
Contents
About the Authors
Abbreviations
List of Figures
List of Tables
1: Introduction
	1.1 What Are Viruses
	1.2 Organisation of a Virus
	1.3 Classification of Viruses
	1.4 How Are Viruses Studied
	1.5 Basic Organisation of a Cell
	1.6 Application of Virology
		1.6.1 Research
		1.6.2 Diagnostics
	1.7 Scope of This Book
	References
2: Isolation of Nucleic Acids
	2.1 Steps Involved in the Isolation of Nucleic Acids
	2.2 Materials
	2.3 Isolation of DNA/RNA
		2.3.1 Isolation of DNA from Cells/Fresh Tissues
		2.3.2 Isolation of RNA from Cells/Fresh Tissues
		2.3.3 Isolation of DNA/RNA from Formalin-Fixed and Paraffin-Embedded Tissues
		2.3.4 Isolation of Subcellular DNA/RNA
	2.4 Determining the Quality and Concentration of Purified Nucleic Acids
	2.5 Reverse Transcription
	2.6 Troubleshooting
	References
3: Isolation of Proteins
	3.1 Steps Involved in the Isolation of Proteins
	3.2 Materials
	3.3 Isolation of Proteins
		3.3.1 Isolation of Whole Cell Proteins
		3.3.2 Isolation of Subcellular Proteins
	3.4 Determination of Protein Purity and Concentration
	3.5 Troubleshooting
	References
4: PCR-Based Techniques
	4.1 Types of PCR
		4.1.1 Standard PCR
		4.1.2 Reverse Transcriptase PCR
		4.1.3 One-Step PCR
		4.1.4 Quantitative PCR
		4.1.5 Multiplex PCR
		4.1.6 Nested PCR
	4.2 Materials
	4.3 Procedure
		4.3.1 PCR Amplification
		4.3.2 Visualisation of PCR Amplicon
		4.3.3 Agarose Gel Electrophoresis
	4.4 Troubleshooting
	References
5: Western Blotting
	5.1 Types of Protein Separation Techniques
		5.1.1 Native or Nondenaturing Gel
		5.1.2 Denaturing Gel
	5.2 Materials
	5.3 Procedure
		5.3.1 SDS-Polyacrylamide Gel Electrophoresis
		5.3.2 Visualisation of PAGE and Immunoblotting
	5.4 Troubleshooting
	References
6: Serological Assays
	6.1 Types of ELISA
		6.1.1 Direct ELISA
		6.1.2 Indirect ELISA
		6.1.3 Sandwich ELISA
		6.1.4 Competitive ELISA
	6.2 Materials
	6.3 Procedure
		6.3.1 Immobilising Antigen or Antibody into ELISA Plate
		6.3.2 Conjugating an Antibody or Antigen
		6.3.3 Detection of Viral Antigen or Antibody
		6.3.4 Signal Detection
	6.4 Troubleshooting
	References
7: Immunoprecipitation
	7.1 Types of Immunoprecipitation
		7.1.1 Individual Protein Immunoprecipitation
		7.1.2 Chromatin Immunoprecipitation
		7.1.3 RNA Immunoprecipitation
		7.1.4 Co-Immunoprecipitation
	7.2 Materials
	7.3 Procedure
		7.3.1 Lysate Preparation
		7.3.2 Antibody Preparation
		7.3.3 Immunoprecipitation
	7.4 Troubleshooting
	References
8: Small Interfering RNA
	8.1 Biogenesis and Mechanism of Action of miRNA
	8.2 Types of siRNA Transfection Techniques
		8.2.1 Viral-Based Transfection Technique
		8.2.2 Lipid-Based siRNA Transfection Technique
		8.2.3 Non-lipid Organic-Based siRNA Technique
		8.2.4 Inorganic siRNA Technique
	8.3 Materials
	8.4 Procedure
		8.4.1 Design and Preparation of siRNA
		8.4.2 Transfection of siRNA into Cells and Gene Silencing
	8.5 Troubleshooting
	References
9: Histological Methods
	9.1 Types of Histological Techniques
	9.2 Materials
	9.3 Histological Sample Preparation
		9.3.1 Cytospin
		9.3.2 Cryostat
		9.3.3 Agarose-Blocked Cells
		9.3.4 Formalin-Fixed, Paraffin-Embedded Tissues
	9.4 Procedure
		9.4.1 Processing of FFPE Blocks
		9.4.2 Antigen Detection
		9.4.3 Signal Detection and Visualisation
	9.5 Troubleshooting
	References
10: Bioinformatics and In Silico Stimulations
	10.1 Sequencing and Sequence Analysis
	10.2 Genetic Relatedness and Phylogenetic Analysis
	10.3 Macromolecules Interactions
		10.3.1 Structural Predictions
		10.3.2 In Silico Macromolecular Interactions
	10.4 Materials
	10.5 Procedure
		10.5.1 Sanger Sequencing Technique
			10.5.1.1 Classical Sanger Sequencing Method
				Preparation of Sequencing Mixture
				Sequencing Thermocycling
				Analysis and Determination of DNA Sequence
			10.5.1.2 Automated Sanger Sequencing Method
				Preparation of Sequencing Mixture
				Sequencing Thermocycling
				Analysis and Determination of DNA Sequence
		10.5.2 Next-Generation Sequencing Technique
		10.5.3 Sequence Analysis
			10.5.3.1 Steps of Blasting and Retrieving Sequences from NCBI Website
			10.5.3.2 Steps in Using the NCBI Website to Search and Retrieve Sequences
			10.5.3.3 Steps of Sequence and Phylogenetic Analyses in BioEdit Software
	10.6 Troubleshooting
	References
11: Summary and Conclusion
	11.1 Summary
	11.2 Conclusion
	References
Index