Wan Wing Kuen. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 114-128). / Abstracts in English and Chinese. / Thesis Abstract --- p.i / Dedication --- p.iii / Acknowledgements --- p.iv / List of Figures --- p.xii / List of Tables --- p.xiv / Abbreviations --- p.xv / Chapter CHAPTER 1: --- INTRODUCTION --- p.1 / Chapter 1.1 --- Research Initiative and Significance --- p.1 / Chapter 1.2 --- Cardiomyocyte and Terminal differentiation --- p.4 / Chapter 1.3 --- Hypertension and Myocardial Hypertrophy --- p.7 / Chapter 1.3.1 --- Hypertension --- p.7 / Chapter 1.3.2 --- Myocardial Hypertrophy --- p.8 / Chapter 1.4 --- Experimental Animal Models --- p.13 / Chapter 1.4.1 --- Spontaneously Hypertensive Rats --- p.15 / Chapter 1.4.2 --- Wistar-Kyoto Rats --- p.16 / Chapter 1.5 --- Combination of Suppression Subtractive Hybridization and cDNA Microarray Analysis --- p.17 / Chapter 1.5.1 --- Suppression Subtractive Hybridization --- p.17 / Chapter 1.5.2 --- cDNA Microarray A nalysis --- p.21 / Chapter 1.5.3 --- Combination of SSH and cDNA microarray --- p.24 / Chapter CHAPTER 2: --- MATERIALS AND METHODS --- p.25 / Chapter 2.1 --- Experimental Animal Models --- p.25 / Chapter 2.2 --- RNA Isolation from Rat Ventricle --- p.26 / Chapter 2.2.1 --- Total RNA Isolation --- p.26 / Chapter 2.2.2 --- Deoxyribonuclease I Digestion --- p.27 / Chapter 2.3 --- Suppression Subtractive Hybridization --- p.29 / Chapter 2.3.1 --- First-Strand cDNA Synthesis --- p.29 / Chapter 2.3.2 --- Second-Strand cDNA Synthesis --- p.30 / Chapter 2.3.3 --- Column Chromatography --- p.31 / Chapter 2.3.4 --- Rsa I Endonuclease Digestion --- p.32 / Chapter 2.3.5 --- Adaptor Ligation --- p.33 / Chapter 2.3.6 --- Ligation Efficiency Analysis --- p.34 / Chapter 2.3.7 --- First Hybridization --- p.35 / Chapter 2.3.8 --- Second Hybridization --- p.36 / Chapter 2.3.9 --- Primary PCR Amplification --- p.36 / Chapter 2.3.10 --- Secondary PCR Amplification --- p.38 / Chapter 2.3.11 --- Subtraction Efficiency Analysis --- p.38 / Chapter 2.4 --- Construction of Subtracted cDNA Libraries --- p.40 / Chapter 2.5 --- cDNA Microarray Analysis --- p.42 / Chapter 2.5.1 --- PCR amplification of Subtracted Clones --- p.42 / Chapter 2.5.2 --- Purification of PCR Products of Subtracted Clones --- p.42 / Chapter 2 5.3 --- Rearrangement of Subtracted Clones into cDNA Microarray Format --- p.43 / Chapter 2.5.4 --- cDNA Microarray Fabrication --- p.43 / Chapter 2.5.5 --- Probe Preparation --- p.44 / Chapter 2.5.6 --- cDNA Microarray Hybridization --- p.46 / Chapter 2.5.7 --- Scanning cDNA Microarray Image and Data Analysis --- p.46 / Chapter 2.6 --- Sequencing of Differentially Expressed Genes --- p.48 / Chapter 2.6.1 --- Dye-terminator Cycle Sequencing --- p.48 / Chapter 2.6.2 --- Post-reaction Cleanup --- p.49 / Chapter 2.6.3 --- Signal Detection and Data Collection --- p.49 / Chapter 2.6.4 --- Sequencing Analysis --- p.50 / Chapter 2.7 --- Reverse Transcription Polymerase Chain Reaction --- p.51 / Chapter 2.8 --- Northern Blot Analysis --- p.54 / Chapter 2.8.1 --- RNA Transfer --- p.54 / Chapter 2.8.2 --- Probe Labeling --- p.55 / Chapter 2.8.3 --- Hybridization --- p.55 / Chapter 2.8.4 --- Chemiluminescent Detection --- p.56 / Chapter CHAPTER 3: --- RESULTS --- p.58 / Chapter 3.1 --- Suppression Subtractive Hybridization --- p.58 / Chapter 3.1.1 --- The Optimal Cycle for SMART cDNA Synthesis --- p.58 / Chapter 3.1.2 --- Adaptor Ligation Efficiency --- p.60 / Chapter 3.1.3 --- Primary and Secondary PCR Amplification of Subtracted cDNA --- p.63 / Chapter 3.1.4 --- Subtraction Efficiency --- p.66 / Chapter 3.2 --- Subtracted cDNA Libraries --- p.68 / Chapter 3.3 --- cDNA Microarray Analysis --- p.69 / Chapter 3.3.1 --- Isolation and Amplification of Subtracted cDNA Clones --- p.69 / Chapter 3.3.2 --- Microarray Scanning and A nalysis --- p.69 / Chapter 3.4 --- Sequencing Results of Subtracted cDNA Clones --- p.81 / Chapter 3.5 --- Reverse Transcription Polymerase Chain Reaction --- p.87 / Chapter 3.6 --- Northern Blot Hybridization --- p.90 / Chapter CHAPTER 4: --- DISCUSSION --- p.93 / Chapter 4.1 --- Subtraction Quality --- p.93 / Chapter 4.2 --- Differential Screening by cDNA Microarray --- p.95 / Chapter 4.2.1 --- Elimination of False Subtracted Clones --- p.95 / Chapter 4.2.2 --- Limitations of cDNA Microarray --- p.96 / Chapter 4.3 --- Differentially Expressed Genes in Hypertensive Heart --- p.98 / Chapter 4.3.1 --- Candidate Genes Showing Up-regulation --- p.99 / Chapter 4.3.1.1 --- Voltage-dependent Anion Channel 1 --- p.99 / Chapter 4.3.1.2 --- Protein Tyrosine Phosphatase 4a 1 --- p.101 / Chapter 4.3.1.3 --- Choline Transporter-like Protein 1 Splice Variant a --- p.102 / Chapter 4.3.2 --- Candidate Genes Showing Down-regulation --- p.103 / Chapter 4.3.2.1 --- Ryanodine Receptor 2 --- p.103 / Chapter 4.3.2.2 --- Guanine Nucleotide-binding Protein β1 Subunit --- p.104 / Chapter 4.3.2.3 --- Solute Carrier Family 3 Member 1 --- p.105 / Chapter 4.4 --- The Pros and Cons of Using Ventricular Tissue but not Cardiomyocytes --- p.107 / Chapter 4.5 --- Future Prospect --- p.109 / Chapter 4.5.1 --- Expression Profiling of Candidate Genes at Different Stages --- p.109 / Chapter 4.5.2 --- In vitro Studies of Candidate Genes --- p.110 / Chapter 4.5.2.1 --- Over-expression of up-regulated genes in Normal Cardiac Cells --- p.110 / Chapter 4.5.2.2 --- Suppression of down-regulated genes in Normal Cardiac Cells --- p.111 / Chapter 4.5.3 --- In vivo Studies of Up-regulated Genes --- p.111 / Chapter 4.5.4 --- Confirmation of Other Potential Candidate Genes --- p.112 / Chapter 4.6 --- Conclusion --- p.113 / REFERENCES --- p.114
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_324446 |
| Date | January 2003 |
| Contributors | Wan, Wing Kuen., Chinese University of Hong Kong Graduate School. Division of Biochemistry. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, xvii, 128 leaves : ill. ; 30 cm. |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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