Basic Science Methods for Clinical Researchers

Basic Science Methods for Clinical Researchers addresses the specific challenges faced by clinicians without a conventional science background. The aim of the book is to introduce the reader to core experimental methods commonly used to answer questions in basic science research and to outline their relative strengths and limitations in generating conclusive data.

This book will be a vital companion for clinicians undertaking laboratory-based science. It will support clinicians in the pursuit of their academic interests and in making an original contribution to their chosen field. In doing so, it will facilitate the development of tomorrow’s clinician scientists and future leaders in discovery science.

• Serves as a helpful guide for clinical researchers who lack a conventional science background
• Organized around research themes pertaining to key biological molecules, from genes, to proteins, cells, and model organisms
• Features protocols, techniques for troubleshooting common problems, and an explanation of the advantages and limitations of a technique in generating conclusive data
• Appendices provide resources for practical research methodology, including legal frameworks for using stem cells and animals in the laboratory, ethical considerations, and good laboratory practice (GLP)

Biomedical researchers; residents and medical students; clinical researchers and students in various medical areas as well as pharmaceutical science and bioengineering

Chapter 1. The Polymerase Chain Reaction: PCR, qPCR, and RT-PCR

• Abstract
• Introduction
• In Principle
• Analysis of the PCR Product
• Phases of PCR
• Variations of PCR Method: Quantitative PCR
• Variations of PCR Method: Reverse Transcription PCR
• In Practice
• Extraction of Total RNA
• Synthesis of cDNA by Reverse Transcription
• Real-Time PCR Amplification
• Data Analysis: Generating a Standard Curve
• Relative Quantification (Pfaffl Method)
• Applications of PCR
• Scenario
• Key Limitations
• Troubleshooting
• Conclusion
• References
• Suggested Further Reading
• Glossary
• List of Acronyms and Abbreviations

Chapter 2. Methods of Cloning

• Abstract
• Introduction
• In Principle
• In Practice
• Applications
• Scenario
• Key Limitations
• Troubleshooting
• Conclusions
• Acknowledgments
• References
• Glossary

Chapter 3. Whole-Mount In Situ Hybridization and a Genotyping Method on Single Xenopus Embryos

• Abstract
• Introduction
• In Principle
• In Practice—Xenopus Whole-Mount In Situ Protocol—Baskets
• Applications
• Key Limitations
• Conclusion
• References
• List of Acronyms and Abbreviations

Chapter 4. Microarrays: An Introduction and Guide to Their Use

• Abstract
• Introduction
• In Principle
• In Practice
• Applications
• Scenario
• Key Limitations
• Troubleshooting
• Conclusion
• References
• Suggested Further Reading
• Glossary
• List of Acronyms and Abbreviations

Chapter 5. Analysis of Human Genetic Variations Using DNA Sequencing

• Abstract
• Introduction
• In Principle
• In Practice
• Applications
• Scenario
• Key Limitations
• Common Trouble Shooting for Sanger Sequencing and NGS
• Conclusion
• References

Chapter 6. Western Blot

• Abstract
• Introduction
• In Principle
• In Practice
• Applications
• Scenario
• Key Limitations
• Troubleshooting
• Conclusion
• References
• Suggested Further Reading
• Glossary

Chapter 7. The Enzyme-Linked Immunosorbent Assay: The Application of ELISA in Clinical Research

• Abstract
• Introduction
• In Principle
• In Practice
• Applications
• Scenario
• Key Limitations
• Troubleshooting
• Conclusion
• References
• Suggested further reading
• Glossary
• List of Acronyms and Abbreviations

Chapter 8. Immunofluorescence

• Abstract
• Introduction
• In Principle
• Direct Immunofluorescence
• Indirect Immunofluorescence
• In Practice
• Applications
• Scenario
• Key Limitations
• Troubleshooting
• Conclusions
• References
• Suggested Further Reading
• Glossary
• LIST OF ACRONYMS AND Abbreviations

Chapter 9. Cell Culture: Growing Cells as Model Systems In Vitro

• Abstract
• Introduction
• In Principle
• In Practice
• Applications
• Scenario
• Key Limitations
• Troubleshooting
• Conclusion
• References
• Glossary
• List of acronyms and Abbreviations

Chapter 10. Flow Cytometry

• Abstract
• Introduction
• In Principle
• Fluidics System: Cell Transport to the Laser Beam
• Optics System: Lasers and Lenses
• Electronics System: Converting the Light Signals into Numerical Data
• In Practice
• Applications
• Scenario
• Limitations of Flow Cytometry
• Troubleshooting
• Conclusions
• References
• Suggested Further reading
• Glossary
• List of Acronyms and Abbreviations

Chapter 11. Transfection

• Abstract
• Introduction
• In Principle
• Chemical Methods
• Physical Methods
• Biological Methods
• In Practice
• Applications
• Scenario
• Key Limitations
• Conclusion
• Acknowledgments
• References
• Glossary

Chapter 12. In Vivo Animal Modeling: Drosophila

• Abstract
• Introduction
• In Principle
• In Practice
• Applications
• Scenario—Human Disease Gene Studies
• Key Limitations
• Troubleshooting
• Conclusion
• Acknowledgments
• References
• SUGGESTED FURTHER READING
• Glossary
• List of Acronyms and Abbreviations

Chapter 13. Zebrafish as a Research Organism: Danio rerio in Biomedical Research

• Abstract
• Introduction
• Use as a Research Organism
• In Principle
• Reproduction
• Development
• Functional Anatomy
• In Practice
• Fish Husbandry
• Procedures On Fish
• Humane Killing
• Genetic Manipulation
• Transgenic Construction
• Gene Activation
• CRISPR-Based Genome Editing
• Physical Manipulation
• Genetic Screens
• Applications
• Scenario—The Innate Immune Response to Cancer Resection
• Key Limitations
• Troubleshooting
• Conclusion
• Acknowledgments
• References
• Suggested Further Reading
• Glossary

Chapter 14. Xenopus as a Model Organism for Biomedical Research

• Abstract
• Introduction
• Use as a Research Organism
• In Principle
• In Practice
• Fertilization and Embryo Manipulation
• Applications
• Key Limitations
• Troubleshooting
• Conclusion
• Acknowledgment
• References
• Suggested Further Reading
• Glossary
• List of Acronyms and Abbreviations

Chapter 15. Basic Mouse Methods for Clinician Researchers: Harnessing the Mouse for Biomedical Research

• Abstract
• Introduction
• In Principle
• In Practice: Basic Mouse Methods
• In Practice: Advanced in vivo Modeling in the Mouse
• Applications of Mouse Models
• Conclusions
• References
• Suggested Further Reading
• Glossary

Appendix A. Legal Framework on the Scientific Use of Animals in Research

• An Introduction to Animal Research
• Regulation
• Ethical Considerations
• Alternatives to Animal Models
• Conclusion
• References

Appendix B. Regulatory Frameworks for Stem Cell Research

• Introduction
• Who Makes the Rules?
• Advances and Corresponding Legislation
• United Kingdom
• Resources for Researchers
• Conclusion
• References

Appendix C. Using Multiple Experimental Methods to Address Basic Science Research Questions

• Introduction
• Why Is Determining Protein Function Important?
• Analyzing Protein X’s Function
• Investigating the Subcellular Localization of Protein X
• Genome-Wide Microarray