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Improvements on quantitative and qualitative analysis of fetal nucleic acids in maternal plasma.

Lo, Yin Wai Wyatt. / "December 2010." / Thesis (M.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 186-206). / Abstracts in English and Chinese. / ABSTRACT --- p.II / 摘要 --- p.VII / ACKNOWLEDGEMENTS --- p.X / PUBLICATIONS --- p.XI / TABLE OF CONTENTS --- p.XII / LIST OF TABLES --- p.XVIII / LIST OF FIGURES --- p.XXI / LIST OF ABBREVIATIONS --- p.XXIV / Chapter SECTION I: --- BACKGROUND --- p.1 / Chapter CHAPTER 1: --- PRENATALTESTNG --- p.2 / Chapter 1.1. --- THE AIM --- p.2 / Chapter 1.2. --- INVASIVE PRENATAL DIAGNOSIS --- p.4 / Chapter 1.3. --- NONINVASIVE PRENATAL SCREENING --- p.6 / Chapter CHAPTER 2: --- NONINVASIVE PRENATAL DIAGNOSIS --- p.10 / Chapter 2.1. --- CIRCULATING FETAL CELLS IN PRENATAL DIAGNOSIS --- p.10 / Chapter 2.2. --- CIRCULATING FETAL NUCLEIC ACIDS IN PRENATAL DIAGNOSIS --- p.12 / Chapter 2.3.1. --- Biology of circulating fetal DNA . --- p.14 / Chapter 2.3.2. --- Clinical applications of circulating fetal DNA --- p.15 / Chapter 2.3.2.1. --- Qualitative analysis of fetal DNA in maternal plasma --- p.16 / Chapter 2.3.2.2. --- Quantitative analysis of fetal DNA in maternal plasma --- p.17 / Chapter 2.4. --- CIRCULATING FETAL RNA IN MATERNAL PLASMA --- p.20 / Chapter 2.4.1. --- Biology of circulating fetal RNA --- p.20 / Chapter 2.4.2. --- Clinical applications of circulating fetal RNA --- p.22 / Chapter 2.4.2.1. --- Quantitative analysis of fetal RNA in maternal plasma --- p.22 / Chapter CHAPTER 3: --- TECHNICAL CHALLENGES IN ANALYZING CIRCULATING FETAL NUCLEIC ACIDS --- p.26 / Chapter 3.1. --- INTRODUCTION --- p.26 / Chapter 3.2. --- "PREANALYTICAL ISSUES IN MATERNAL PLASMA NUCLEIC ACID ANALYSE"";" --- p.28 / Chapter 3.2.1. --- Low abundance of cell-free fetal nucleic acids in maternal plasma --- p.28 / Chapter 3.2.2. --- High level of maternal background in maternal plasma --- p.30 / Chapter 3.3. --- ANALYTICAL ISSUES IN MATERNAL PLASMA NUCLEIC ACID ANALYS --- p.IS / Chapter 3.3.1. --- Imprecise measurement of fetal nucleic acid quantity --- p.33 / Chapter 3.3.2. --- Coexistence of fetal nucleic acids and maternal nucleic acid background in maternal plasma --- p.36 / Chapter 3.4. --- AIMS OF THIS THESIS --- p.41 / Chapter SECTION II: --- MATERIALS AND METHODS --- p.42 / Chapter CHAPTER 4: --- QUANTITATIVE AND QUALITATIVE ANALYSIS OF NUCLEIC ACIDS --- p.43 / Chapter 4.1. --- SAMPLE COLLECTION AND PROCESSING --- p.43 / Chapter 4.1.1. --- Preparation of plasma and blood cells --- p.43 / Chapter 4.1.2. --- Preparation of placental tissues --- p.44 / Chapter 4.2. --- NUCLEIC ACID EXTRACTION --- p.45 / Chapter 4.2.1. --- Extraction of total RNA --- p.45 / Chapter 4.2.1.1. --- Plasma samples --- p.45 / Chapter 4.2.1.2. --- Placental tissue samples --- p.46 / Chapter 4.2.2. --- Extraction of genomic DNA --- p.48 / Chapter 4.2.2.1. --- Plasma samples --- p.48 / Chapter 4.2.2.2. --- Blood cell samples --- p.48 / Chapter 4.3. --- CONVENTIONAL REAL-TIME PCR ANALYSIS OF NUCLEIC ACIDS --- p.50 / Chapter 4.3.1. --- Principles of real-time polymerase chain reaction --- p.50 / Chapter 4.3.2. --- Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) --- p.52 / Chapter 4.3.3. --- Quantitative polymerase chain reaction (qPCR) --- p.53 / Chapter 4.4. --- DIGITAL REAL-TIME PCR ANALYSIS OF NUCLEIC ACIDS --- p.55 / Chapter 4.4.1. --- Principles of digital PCR (dPCR) --- p.55 / Chapter 4.4.2. --- 384-reaction well dPCR v --- p.56 / Chapter 4.4.3. --- Microfluidics dPCR --- p.57 / Chapter 4.5. --- MATRIX-ASSISTED LASER DESORPTIONIONIZATION/TIME-OF-FLIGHT MASS SPECTROMETRY (MALDI-TOF MS) ANALYSIS OF NUCLEIC ACIDS --- p.59 / Chapter 4.5.1. --- Principles of MALDI-TOF MS --- p.59 / Chapter 4.5.2. --- DNA genotyping analysis by MassArray Homogenous MassExtend (hME) assay --- p.60 / Chapter 4.6. --- CLONING AND DNA SEQUENCING --- p.63 / Chapter SECTION III: --- IMPROVEMENTS ON MATERNAL PLASMA ANALYSIS OF CIRCULATING RNA --- p.65 / Chapter CHAPTER 5: --- ENRICHMENT OF PLACENTA EXPRESSED MRNA MARKERS BY WHOLE TRANSCRIPTOME PREAMPLIFICATION --- p.66 / Chapter 5.1. --- INTRODUCTION --- p.66 / Chapter 5.2. --- MATERIALS AND METHODS --- p.69 / Chapter 5.2.1. --- Study design --- p.69 / Chapter 5.2.2. --- Subjects and sample collection --- p.71 / Chapter 5.2.3. --- RNA extraction and sample dilution --- p.71 / Chapter 5.2.4. --- Preamplification --- p.73 / Chapter 5.2.5. --- qPCR analysis --- p.74 / Chapter 5.3. --- RESULTS --- p.83 / Chapter 5.3.1. --- Comparison of mRNA expression profiles in placental tissues with and without preamplification --- p.83 / Chapter 5.3.1.1. --- Undiluted placental tissue RNA --- p.84 / Chapter 5.3.1.2. --- Diluted placental tissue RNA --- p.88 / Chapter 5.3.2. --- The effect of RNA input on the degree of amplification --- p.92 / Chapter 5.3.2.1. --- Correlation between RNA input and RNA output --- p.94 / Chapter 5.3.2.2. --- Correlation between RNA input and output/input ratio --- p.96 / Chapter 5.3.3. --- Preamplification of maternal plasma RNA --- p.98 / Chapter 5.3.3.1. --- Concentrations of placenta expressed mRNA in third trimester maternal plasma --- p.98 / Chapter 5.3.3.2. --- Concentrations of placenta expressed mRNA in first trimester maternal plasma --- p.100 / Chapter 5.4. --- DISCUSSION --- p.102 / Chapter SECTION IV: --- IMPROVEMENTS ON MATERNAL PLASMA ANALYSIS OF CIRCULATING DNA --- p.105 / Chapter CHAPTER 6: --- ACCURATE GENE DOSAGE ANALYSIS BY MULTIPLEX QPCR --- p.106 / Chapter 6.1. --- INTRODUCTION --- p.106 / Chapter 6.2. --- MATERIALS AND METHODS --- p.110 / Chapter 6.2.1. --- Study design --- p.110 / Chapter 6.2.2. --- Subjects and sample collection --- p.112 / Chapter 6.2.3. --- DNA extraction and sample dilution --- p.113 / Chapter 6.2.4. --- qPCR analysis --- p.113 / Chapter 6.2.4.1. --- Monoplex assays --- p.114 / Chapter 6.2.4.2. --- Multiplex assays --- p.114 / Chapter 6.2.5. --- Microfluidics dPCR assay --- p.122 / Chapter 6.2.6. --- Gene Dosage Comparison --- p.122 / Chapter 6.2.6.1. --- In adult male samples --- p.123 / Chapter 6.2.6.2. --- In maternal plasma samples --- p.123 / Chapter 6.3. --- RESULTS --- p.125 / Chapter 6.3.1. --- The influence of using the same and different sets of primers for amplifying different chromosomal loci --- p.125 / Chapter 6.3.2. --- Effects of using monoplex and multiplex real-time PCR formulations --- p.130 / Chapter 6.3.3. --- Effects of incorporating calibration curves for template quantification in conventional qPCR --- p.135 / Chapter 6.4. --- DISCUSSION --- p.140 / Chapter CHAPTER 7: --- DPCR DETECTION OF PATERNALLY INHERITED POINT MUTATIONS --- p.144 / Chapter 7.1. --- INTRODUCTION --- p.144 / Chapter 7.2. --- MATERIALS AND METHODS --- p.153 / Chapter 7.2.1. --- Study design --- p.153 / Chapter 7.2.2. --- Subjects and sample collection --- p.157 / Chapter 7.2.3. --- DNA extraction and sample preparation --- p.158 / Chapter 7.2.4. --- MassArray hME assays --- p.159 / Chapter 7.2.5. --- dPCR assay --- p.159 / Chapter 7.3. --- RESULTS --- p.161 / Chapter 7.3.1. --- Validation of the digital HbE assay --- p.161 / Chapter 7.3.2. --- Determination of the minimum fetal DNA amount required for digital PCR detection --- p.165 / Chapter 7.3.3. --- Detection of paternally inherited fetal HbE mutation in maternal plasma --- p.172 / Chapter 7.4. --- DISCUSSION --- p.175 / Chapter SECTION V: --- CONCLUDING REMARKS --- p.180 / Chapter CHAPTER 8: --- CONCLUSION AND FUTURE PERSPECTIVES --- p.181 / Chapter 8.1. --- IMPROVEMENTS ON QUANTITATIVE AND QUALITATIVE ANALYSIS OF FETAL NUCLEIC ACIDS IN MATERNAL PLASMA --- p.181 / Chapter 8.2. --- PERSPECTIVES FOR FUTURE WORK --- p.184 / REFERENCES --- p.186

Identiferoai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_327444
Date January 2011
ContributorsLo, Yin Wai Wyatt., Chinese University of Hong Kong Graduate School. Division of Chemical Pathology.
Source SetsThe Chinese University of Hong Kong
LanguageEnglish, Chinese
Detected LanguageEnglish
TypeText, bibliography
Formatprint, xxv, 206 leaves : ill. ; 30 cm.
RightsUse 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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