Shing, Ka Fai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 158-188). / Abstracts in English and Chinese. / ABSTRACT --- p.i / 摘要 --- p.iv / ACKNOWLEDGEMENTS --- p.vi / TABLE OF CONTENTS --- p.vii / LIST OF TABLES --- p.x / LIST OF FIGURES --- p.xii / Chapter SECTION I: --- BACKGROUND --- p.1 / Chapter CHAPTER 1: --- CELL-FREE NUCLEIC ACIDS IN HUMAN BODILY FLUIDS --- p.2 / Chapter 1.1 --- Early studies on the presence of cell-free nucleic acids in human bodily fluids --- p.2 / Chapter 1.2 --- Circulating nucleic acids in plasma and serum --- p.2 / Chapter 1.2.1 --- Cancer Detection --- p.3 / Chapter 1.2.1.1 --- Circulating tumor-derived DNA --- p.3 / Chapter 1.2.1.2 --- Circulating tumor-derived RNA --- p.5 / Chapter 1.2.2 --- Prenatal diagnosis --- p.7 / Chapter 1.2.2.1 --- Circulating fetal DNA --- p.7 / Chapter 1.2.2.2 --- Circulating fetal messenger RNA --- p.11 / Chapter 1.2.2.3 --- Circulating placental microRNA --- p.13 / Chapter 1.3 --- Cell-free nucleic acids in urine --- p.14 / Chapter 1.3.1 --- Transrenal DNA (Tr-DNA) --- p.15 / Chapter 1.3.1.1 --- Biology of Tr-DNA --- p.15 / Chapter 1.3.1.2 --- Detection of fetal-derived Tr-DNA --- p.15 / Chapter 1.3.1.3 --- Potential problems associated with the detection of Tr-DNA --- p.16 / Chapter 1.3.2 --- Cell-free DNA in urine as released from the urinary tract --- p.17 / Chapter 1.4 --- Other bodily fluids with cell-free nucleic acids --- p.18 / Chapter 1.4.1 --- Amniotic fluid --- p.19 / Chapter 1.4.2 --- Cerebrospinal fluid (CSF) --- p.20 / Chapter 1.4.3 --- Peritoneal fluid --- p.20 / Chapter CHAPTER 2: --- MICRORNA IN HUMANS --- p.21 / Chapter 2.1 --- Introduction --- p.21 / Chapter 2.2 --- Biogenesis --- p.21 / Chapter 2.2.1 --- Transcription of microRNA genes --- p.21 / Chapter 2.2.2 --- Processing and maturation of microRNA precursors --- p.23 / Chapter 2.3 --- Mechanisms of gene regulation --- p.24 / Chapter 2.3.1 --- Cleavage of target mRNA --- p.24 / Chapter 2.3.2 --- Translational repression of mRNA --- p.25 / Chapter 2.4 --- Functional roles of microRNAs --- p.25 / Chapter 2.4.1 --- Oncogenesis --- p.25 / Chapter 2.4.2 --- Programmed cell death --- p.26 / Chapter 2.4.3 --- Cellular differentiation and development --- p.27 / Chapter 2.4.4 --- Regulation of physiological and cellular processes --- p.28 / Chapter 2.5 --- Aim of this thesis --- p.28 / Chapter SECTION II: --- MATERIALS AND METHODS --- p.30 / Chapter CHAPTER 3: --- QUANTITATIVE ANALYSIS OF CIRCULATING AND URINARY NUCLEIC ACIDS --- p.31 / Chapter 3.1 --- Preparation of samples --- p.31 / Chapter 3.1.1 --- Preparation of plasma --- p.31 / Chapter 3.1.2 --- Preparation of blood cells --- p.32 / Chapter 3.1.3 --- Preparation of placental tissue --- p.32 / Chapter 3.1.4 --- Preparation of urine and urine cell pellet --- p.32 / Chapter 3.2 --- Nucleic acid extraction --- p.33 / Chapter 3.2.1 --- "Extraction of small RNA-containing total RNA from plasma, blood cells and placental tissue" --- p.33 / Chapter 3.2.2 --- Extraction of DNA from urine --- p.37 / Chapter 3.3 --- Quantitative measurements of nucleic acids --- p.38 / Chapter 3.3.1 --- Principle of real-time quantitative PCR --- p.38 / Chapter 3.3.2 --- One-step QRT-PCR assays for mRNA quantification --- p.40 / Chapter 3.3.2.1 --- Principle --- p.40 / Chapter 3.3.2.2 --- Quantification of human placental lactogen （hPL) mRNA --- p.40 / Chapter 3.3.3 --- Two-step QRT-PCR assays for microRNA quantification --- p.45 / Chapter 3.3.3.1 --- Principle --- p.45 / Chapter 3.3.3.2 --- Advantages --- p.46 / Chapter 3.3.3.3 --- TaqMan® MicroRNA Assays --- p.47 / Chapter 3.3.4 --- QPCR assays for DNA quantification --- p.53 / Chapter 3.3.4.1 --- Principle --- p.53 / Chapter 3.3.4.2 --- Quantification of the leptin gene and the sex-determining region on Ychromosome gene --- p.53 / Chapter 3.4 --- Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) --- p.57 / Chapter 3.4.1 --- Principle --- p.57 / Chapter 3.4.2 --- Zinc finger protein gene assay for determining the fractional concentration of male DNA --- p.58 / Chapter 3.5 --- Statistical analyses --- p.65 / Chapter SECTION III: --- CIRCULATING PLACENTAL MICRORNAS IN MATERNAL PLASMA AS MARKERS FOR PRENATAL DIAGNOSIS --- p.66 / Chapter CHAPTER 4: --- THE EXISTENCE AND QUANTITATIVE DETECTION OF CELL-FREE MICRORNAS IN PLASMA --- p.67 / Chapter 4.1 --- Introduction --- p.67 / Chapter 4.2 --- Materials and methods --- p.69 / Chapter 4.2.1 --- Sample collection --- p.69 / Chapter 4.2.2 --- Experimental design --- p.69 / Chapter 4.2.3 --- RNA extraction and quantification --- p.72 / Chapter 4.3 --- Results --- p.75 / Chapter 4.3.1 --- Validation of two-step QRT-PCR system for miRNA quantification --- p.75 / Chapter 4.3.2 --- Detection of cell-free miRNA in maternal plasma --- p.82 / Chapter 4.4 --- Discussion --- p.82 / Chapter CHAPTER 5: --- SYSTEMATIC IDENTIFICATION AND CHARACTERIZATION OF PLACENTAL MICRORNAS IN MATERNAL PLASMA --- p.86 / Chapter 5.1 --- Introduction --- p.86 / Chapter 5.2 --- Materials and methods --- p.88 / Chapter 5.2.1 --- Sample collection --- p.88 / Chapter 5.2.2 --- Experimental design --- p.88 / Chapter 5.2.3 --- RNA extraction and miRNA quantification --- p.91 / Chapter 5.3 --- Results --- p.93 / Chapter 5.3.1 --- A systematic search for placental miRNAs in maternal plasma using two-step QRT-PCR assays --- p.93 / Chapter 5.3.2 --- Detection rate and clearance kinetics of placental miRNAs in maternal plasma --- p.97 / Chapter 5.3.3 --- Effects of filtering maternal plasma on the concentration of placental miRNA and mRNA --- p.99 / Chapter 5.3.5 --- Temporal profile of placental miRNA concentrations in maternal plasma across different trimesters of pregnancies --- p.103 / Chapter 5.4 --- Discussion --- p.115 / Chapter SECTION IV: --- DETECTION OF CELL-FREE DNA IN URINE --- p.119 / Chapter CHAPTER 6: --- HEMATOPOIETIC STEM CELL TRANSPLANTATION RECIPIENTS AS A MODEL TO STUDY CELL-FREE DNA IN URINE --- p.120 / Chapter 6.1 --- Introduction --- p.120 / Chapter 6.2 --- Materials and methods --- p.123 / Chapter 6.2.1 --- Sample collection --- p.123 / Chapter 6.2.2 --- Experimental design --- p.124 / Chapter 6.2.3 --- DNA extraction and quantification --- p.125 / Chapter 6.3 --- Results --- p.128 / Chapter 6.3.1 --- Validation of the zinc finger protein gene assay --- p.128 / Chapter 6.3.2 --- Fractional concentration of male DNA in blood cells and plasma of sex-mismatched HSCT patients --- p.129 / Chapter 6.3.3 --- Fractional concentration of male DNA in the urine and the urine cell pellets of sex-mismatched HSCT patients --- p.131 / Chapter 6.3.4 --- Size distribution of cell-free DNA in peripheral blood and urine samples of sex-mismatched HSCT patients --- p.132 / Amplicon size --- p.138 / Chapter 6.4 --- Discussion --- p.143 / Chapter SECTION V: --- CONCLUDING REMARKS --- p.147 / Chapter CHAPTER 7: --- CONCLUSION AND FUTURE PERSPECTIVES --- p.148 / Chapter 7.1 --- Circulating miRNA is a valuable resource for molecular analysis --- p.148 / Chapter 7.2 --- The presence of donor-derived DNA in the urine of HSCT recipients --- p.150 / Chapter 7.3 --- Prospects for future work --- p.152 / APPENDIX 1 --- p.154 / REFERENCES --- p.158
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_326027 |
| Date | January 2007 |
| Contributors | Shing, Ka Fai., Chinese University of Hong Kong Graduate School. Division of Chemical Pathology. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, xiii, 188 leaves : ill. (some col.) ; 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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