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

Lysophosphatidic acid (LPA) influences many physiological processes, such as brain and vascular development. It is associated with several diseases including ovarian cancer, breast cancer, prostate cancer, colorectal cancer, hepatocellular carcinoma, multiple myeloma atherosclerotic diseases, cardiovascular diseases, pulmonary inflammatory diseases and renal diseases. LPA plasma and serum levels have been reported to be important values in diagnosing ovarian cancer and other diseases. However, the extraction and quantification of LPA in plasma are very challenging because of the low physiological concentration and similar structures of LPA to other phospholipids. Many previous studies have not described the separation of LPA from other phospholipids, which may make analyses more challenging than necessary. We developed an SPE extraction method for plasma LPA that can extract LPA at high purity. We also developed an HPLC post-column fluorescence detection method that allows the efficient quantification of LPA. These methods were used in a clinical study for ovarian cancer diagnosis to help validate LPA as a biomarker of ovarian cancer. Moreover, molecular imprinted polymers (MIPs) were designed and synthesized as material for the improved extraction of LPA. Compared to the commercially available materials, the MIP developed shows enhanced selectivity for LPA. The extraction was overall relatively more efficient and less labor-intensive.