Wong, Hoi Kin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 219-235). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.vi / Publications --- p.vii / Abbreviations --- p.viii / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Overview of Human Eye Developmental Process and Structure --- p.1 / Chapter 1.1.1 --- Developmental Process of the Eye --- p.2 / Chapter 1.1.2 --- Anatomy of Human Eye --- p.3 / Chapter 1.1.2.1 --- General Structure and Function of Human Eye --- p.4 / Chapter 1.1.2.2 --- Functional Architecture of Retina --- p.7 / Chapter 1.1.2.2.1 --- Photoreceptors --- p.8 / Chapter 1.1.2.2.2 --- Bipolar cell --- p.9 / Chapter 1.1.2.2.3 --- Horizontal cell --- p.10 / Chapter 1.1.2.2.4 --- Amacrine cell --- p.10 / Chapter 1.1.2.2.5 --- Interplexiform cell --- p.11 / Chapter 1.1.2.2.6 --- Muller glial cell --- p.11 / Chapter 1.1.2.2.7 --- Retinal ganglion cell --- p.12 / Chapter 1.1.3 --- Embryology of Human Eye --- p.12 / Chapter 1.1.3.1 --- Embryogenesis of Retina --- p.13 / Chapter 1.2 --- Identity of Retinal Stem Cells --- p.15 / Chapter 1.3 --- Molecular Mechanism of Early Retinogenesis --- p.17 / Chapter 1.3.1 --- Intrinsic Coding Genes for Retinogenesis --- p.18 / Chapter 1.3.1.1 --- PAX6 --- p.19 / Chapter 1.3.1.2 --- genes family --- p.20 / Chapter 1.3.1.3 --- RAX --- p.21 / Chapter 1.3.1.4 --- Repressor-type bHLH genes --- p.22 / Chapter 1.3.1.5 --- Activator-type bHLH genes --- p.23 / Chapter 1.3.1.6 --- POU4F gene family --- p.24 / Chapter 1.3.1.7 --- DLX1/DLX2 --- p.25 / Chapter 1.3.1.8 --- Others --- p.26 / Chapter 1.3.2 --- Extrinsic Factors for Retinogenesis --- p.27 / Chapter 1.3.2.1 --- Extrinsic morphogens --- p.27 / Chapter 1.3.2.2 --- Notch Signalling Pathway --- p.29 / Chapter 1.3.2.3 --- Sonic Hedgehog Signalling --- p.29 / Chapter 1.3.2.4 --- Wnt Signaling Pathway --- p.30 / Chapter 1.3.2.5 --- Cell Death Signalling during Retinogenesis --- p.31 / Chapter 1.4 --- Non-coding RNAs: MicroRNA --- p.34 / Chapter 1.4.1 --- Biogenesis and Post-Transcriptional Regulation Mechanism of MicroRNA --- p.35 / Chapter 1.4.2 --- Expression of Non-coding Genes in Retina and the Role in Retina Development --- p.37 / Chapter 1.5 --- Human Disorders Related to Retina Defect --- p.38 / Chapter Chapter 2 --- Project Objective and Outline --- p.42 / Chapter Chapter 3 --- Materials and Methods / Chapter 3.1 --- Samples Collection --- p.44 / Chapter 3.1.1 --- Isolation of Retina --- p.45 / Chapter 3.1.2 --- Samples Processing and Storage --- p.46 / Chapter 3.1.3 --- Total RNA extraction --- p.46 / Chapter 3.2 --- RNA Quality Control --- p.47 / Chapter 3.2.1 --- NanoDrop ND-1000 --- p.47 / Chapter 3.2.2 --- Agilent 2100 Bioanalyzer --- p.48 / Chapter 3.3 --- Expression profiling by Microarray Experiments --- p.51 / Chapter 3.3.1 --- Gene Expression Microarray --- p.52 / Chapter 3.3.1.1 --- Prepare Labelling Reaction --- p.52 / Chapter 3.3.1.2 --- Purify the Labelled/Amplified RNA --- p.54 / Chapter 3.3.1.3 --- Prepare Hybridization Samples --- p.55 / Chapter 3.3.1.4 --- "Hybridization, Washing and Scanning" --- p.55 / Chapter 3.3.1.5 --- Gene Expression Data Quality and Acquisition --- p.57 / Chapter 3.3.2 --- MicroRNA Microarray --- p.58 / Chapter 3.3.2.1 --- Dephosphorylation and Labeling Reaction --- p.58 / Chapter 3.3.2.2 --- Gel Filtration to Purify the Labelled RNA --- p.60 / Chapter 3.3.2.3 --- Hybridization Sample Preparation --- p.61 / Chapter 3.3.2.4 --- Prepare the Hybridization Assembly --- p.61 / Chapter 3.3.2.5 --- Washing and scanning --- p.62 / Chapter 3.3.2.6 --- MiRNA Data Quality and Acquisition --- p.62 / Chapter 3.4 --- Statistical Analysis --- p.63 / Chapter 3.4.1 --- Data Normalization --- p.63 / Chapter 3.4.2 --- Significant tests --- p.64 / Chapter 3.4.3 --- Clustering --- p.65 / Chapter 3.5 --- Quantitative Real-Time Polymerase Chain Reaction --- p.65 / Chapter 3.5.1 --- Reverse Transcription --- p.65 / Chapter 3.5.2 --- Quantitative Real-Time PCR --- p.66 / Chapter 3.6 --- Histochemical Staining --- p.67 / Chapter 3.6.1 --- Haematoxylin-and-eosin staining --- p.67 / Chapter 3.6.2 --- Immunohistochemical staining --- p.67 / Chapter 3.6.3 --- In Situ Hybridization (ISH) for MicroRNA --- p.69 / Chapter Chapter 4 --- Results / Chapter 4.1 --- Temporal Gene Expression of Human Fetal Retina --- p.71 / Chapter 4.1.1 --- Tissue specificity by Principal Component Analysis --- p.73 / Chapter 4.1.2 --- mRNA Expression Profiling from Gestational Week 9 to Week 15 --- p.74 / Chapter 4.1.3 --- Characterization of Known Eye Specific Genes in Early Gestation --- p.76 / Chapter 4.2 --- Differential Expressed Genes between Gestational Week 9 and Week 15 --- p.78 / Chapter 4.2.1 --- Unsupervised Clustering between Gestational Week 9 and Week 15 --- p.78 / Chapter 4.2.2 --- Statistical Calculation of Significantly Differential Expressed Gene by Using GeneSpring 7.3.1 --- p.79 / Chapter 4.2.3 --- Statistical Clustering of Unique Expression Profile by Using Self-organizing Map (SOM) Clustering --- p.81 / Chapter 4.2.4 --- Functional Annotation of Differentially Expressed Genes --- p.82 / Chapter 4.3 --- Quantitation of Candidate Eye-Specific Genes by Real-Time PCR --- p.84 / Chapter 4.4 --- Novelty of the human Fetal Retina mRNAs --- p.85 / Chapter 4.5 --- MicroRNA Expression Profiling in Early Retinogenesis --- p.87 / Chapter 4.6 --- Correlation between Coding and Non-coding RNA According to the Expression Pattern on Microarray --- p.90 / Chapter 4.6.1 --- MiRNA Target Gene Analysis --- p.90 / Chapter 4.6.2 --- Correlation analysis between miRNA and its target transcripts according to Global gene expression profile in retina --- p.91 / Chapter Chapter 5 --- Discussion / Chapter 5.1 --- Characterization of the Gene Expression Profile --- p.94 / Chapter 5.1.1 --- Indigenous Genes Expression in Human Fetal Retina --- p.95 / Chapter 5.1.1.1 --- Overview of the mRNA Expression --- p.95 / Chapter 5.1.1.2 --- Characterization by Gene Ontology and Human KEGG Pathway --- p.99 / Chapter 5.1.2 --- Gene Expression Data Comparison from Public Database --- p.101 / Chapter 5.2 --- Stage Specific Genes Expressed on Week 9 and Week 15 --- p.102 / Chapter 5.2.1 --- Characterization of Stage Specific Genes by Gene Ontology Analysis --- p.103 / Chapter 5.2.2 --- Known and new retinal-specific Genes Upregulated on Gestational Week 9 --- p.104 / Chapter 5.2.2.1 --- ALDH1A1A/STRA6 --- p.104 / Chapter 5.2.2.2 --- GAL --- p.105 / Chapter 5.2.2.3 --- CNTN2/SLIT102 --- p.107 / Chapter 5.2.2.4 --- LMINC3 --- p.108 / Chapter 5.2.2.5 --- DLX1/DLX2 --- p.109 / Chapter 5.2.3 --- Genes Upregulated on Gestational Week 15 --- p.110 / Chapter 5.2.3.1 --- RCVN/RCV1 --- p.110 / Chapter 5.2.3.2 --- PAVLB --- p.111 / Chapter 5.2.3.3 --- Presence of Synaptic and Neuronal Signal Transduction Activities in Week 15 Retina --- p.111 / Chapter 5.2.3.4 --- Cell differentiation and Development --- p.112 / Chapter 5.3 --- Expression Characteristic of MicroRNA in Embryonic Retina and the Related Targets --- p.114 / Chapter 5.3.1 --- Other Highly Expressed MiRNAs --- p.115 / Chapter 5.3.2 --- Highly Expressed miRNAs and their Potential Predicted target Function --- p.117 / Chapter 5.3.2.1 --- MiR-124a --- p.117 / Chapter 5.3.3 --- Expression Trends and the Corresponding MiRNAs Analysis --- p.119 / Chapter 5.4 --- Potential Studies for Further Investigation --- p.120 / Chapter Chapter 6 --- Conclusions --- p.122 / Tables and Figures / Figure 1.1 Embryogenesis of early human eye development --- p.124 / Figure 1.2 The anatomy of human eye- --- p.125 / Figure 1.3 Schematic diagram of general retina structure --- p.126 / Figure 1.4 Schematic diagram of photoreceptor cells (cone and rod) --- p.127 / Figure 1.5 The proliferation and differentiation of neural retina between gestational week 6 and week 8 --- p.128 / Figure 1.6 The time line for retinogenesis between the 2nd month and the 6th month of human gestation and the corresponding time line of mouse retinogenesis between the E12 and the Pll --- p.129 / Figure 1.7 Schematic diagram of Notch signaling pathway --- p.130 / Figure 1.8 Summary of the homeobox gene and bHLH gene expression to trigger particular cell type differentiation from retinal progenitor cells --- p.131 / Figure 1.9 Summary of molecular interactions during retinogenesis --- p.132 / Figure 1.10 Signal transduction pathway of sonic hedgehog --- p.133 / Figure 1.11 Biogenesis of microRNA (miRNA) and posttranscriptional regulation of mRNA --- p.134 / Figure 1.12 Summary of the expression of various miRNAs in retina and other neuronal tissues reported in animal studies --- p.135 / Figure 1.13 Number of the mapped loci and the identified retina disease genes between 1980 and 2008 --- p.136 / Figure 3.1 A typical RNA optical density (OD) profile exported from Nanodrop --- p.137 / Figure 3.2 Detection of Cy3 labelled cRNA and the corresponding specificity by Nanodrop ND-1000 --- p.138 / Figure 3.3 RNA quality profile generated from Agilent2100 Bioanalyzer --- p.139 / Figure 3.4 Amplified and fluorescence labelled cRNA profile from Agilent 2100 Bioanalyzer --- p.140 / Figure 3.5 Scatter plot of the fluorescent same fibroblast sample labelled Cy3 and Cy5 separately --- p.141 / Figure 3.6 Scatter plot of 2 identical placenta miRNA separately hybridized on 2 arrays on the same chip --- p.142 / Equation 3.1 Calculation of specificity from fluorescence labelled cRNA samples --- p.143 / Table 3.1 Information of human fetal retina samples involved in this study --- p.144 / Table 3.2 Summary of sample selection criteria by using NanoDrop ND-1000 and Agilent 2100 Bioanalyzer --- p.145 / Figure 4.1 Histogram of expressed probes which express on the gene expression microarrays from gestational week 9 to week 15 --- p.146 / Figure 4.2 Gene ontology (GO) study in nervous system development --- p.147 / Figure 4.3 Gene ontology (GO) study in sensory perception --- p.148 / Figure 4.4 Gene ontology (GO) study in cell growth --- p.149 / Figure 4.5 Gene ontology (GO) study in biological process --- p.150 / Figure 4.6 Box plot of 27 samples from gene expression microarray experiment --- p.151 / "Figure 4.7 Principal component analysis of fetal retina tissues, retinoblastoma tissues and retinoblastoma cell line" --- p.152 / Figure 4.8 Hierarchical clustering of human fetal retina tissues and retinoblastoma samples in gene expression microarray experiments --- p.153 / Figure 4.9 Gene expression profile of human fetal retina from gestational week 9 to week 15 --- p.154 / Figure 4.10 Gene expression profiles of PAX6 and SOX3 --- p.155 / Table 4.3 POU4F1 expression data generated from gene expression microarray from different gestational weeks --- p.156 / Figure 4.11 Graphical illustration of the expression pattern of POU4F1 in retina from gene expression microarray between human gestational week 9 to week 15 --- p.156 / Figure 4.12 Gene expression results of POU4Fl from real-time --- p.157 / Figure 4.13 Retinal ganglion cell differentiation in human fetal retina --- p.158 / Figure 4.14 Hierarchical clustering of human fetal retina tissues comparing gestational week 9 and week 15 with retinoblastoma samples --- p.159 / Figure 4.15 H&E staining of human fetal retina tissues from gestational week 9 to week 15 --- p.160 / Figure 4.16 Scatter plot of differentially expressed genes fold change equal to 2 between gestational week 9 and week 15 --- p.161 / Figure 4.17 Volcano plot of differentially statistical significant expressed genes on gestational week 9 and week 15 --- p.162 / Figure 4.18 A continuous line plot for the differential expressed genes at gestational week 9 and week 15 --- p.163 / Figure 4.19 Self-organizing map clustering (SOM) of the expression library base on the result Figure 4.9 --- p.164 / Figure 4.20 Selected SOM clusters from the Figure 4.22 --- p.165 / Figure 4.21 Gene ontology study of genes commonly expressed in differential expressed gene list (Figure 4.20) and the selected gene clusters in SOM clustering- --- p.166 / "Figure 4.22 Gene ontology diagram for grouping 385 genes differentially expressed on gestational week 15 and commonly found in SOM 226}0بcluster 6,1' with different functions and characteristics" --- p.167 / Figure 4.23 Results comparison between microarray experiment and quantitative real-time PCR validation of temporal expressed genes on gestational week 9 --- p.168 / Figure 4.24 Results comparison between microarray experiment and quantitative real-time PCR validation of temporal expressed genes on gestational week 15 --- p.169 / Figure 4.25 Gene cluster specifically find in this study by compare with RetinaCentral database --- p.170 / Figure 4.26 Histogram of miRNA expression distribution of all 470 miRNAs --- p.171 / Figure 4.27 Hierarchical clustering of miRNA expression array on human fetal retinas and retinoblastoma samples --- p.172 / Figure 4.28 The correlation distribution of miR-124a and mRNAs --- p.173 / Figure 4.29 Expression profiles of GAL and the corresponding- miRNAs searched from TargetScan 4.1 --- p.174 / Figure 4.30 Scatter plot of miR-345 and GAL --- p.175 / Figure 4.31 Expression profiles of DLX1 and the corresponding miRNAs searched from TargetScan 4.2 --- p.176 / Figure 4.32 Expression profiles of DLX2 and the corresponding miRNAs searched from TargetScan 4.1 --- p.177 / Figure 4.33 In situ hybridization (ISH) of miR-182 of human fetal retina on gestational week 9 and week 17 --- p.178 / Figure 4.34 Expression of miR-182 in retinoblastoma tissue --- p.179 / Table 4.1 KEGG pathway analysis from GeneSpring 7.3.1 --- p.180 / Table 4.2 Expression signals of the genes associated with retina development --- p.181 / Table 4.6 Assorted gene ontology terms under the category of molecular function and biological process --- p.182 / Figure 4.7 Selected genes from common gene list on week 9 and week 15 for quantitative real-time PCR --- p.183 / "Table 4.8 Selected genes with expression intensity higher than100,000 with the corresponding UniGene ID, Genbank ID and function(s)" --- p.184 / Table 4.9 The top 25 highly expressed miRNA and the corresponding signal and recent finding on the neuronal organ --- p.188 / Table 4.10 Pathway analysis from GeneSpring shows the related pathway and target genes from the top 10 highly expressed miRNAs --- p.190 / Table 4.11 Genes and miRNA with expression correlation coefficient smaller than -0.7 and gene expression signal higher than / Figure 5.1 Summary of pathway interactions from KEGG pathway analysis --- p.193 / Figure 5.2 Expression tread of ALDHlAl normalized by control signals --- p.194 / Figure 5.3 The retinoic acid metabolize pathway between egg yolk and placenta --- p.195 / Figure 5.4 The expression of other genes involved in retinoic acid metabolic pathway --- p.196 / Figure 5.5 Expression signal of various galanin receptors in our data --- p.197 / Figure 5.6 Expression signal of CNTN2 from gestational week 9 to week 15 --- p.198 / Figure 5.7 Expression of DLX1 and DLX2 and the corresponding correlation --- p.199 / Figure 5.8 Expression trend of RCVN from gestational week 9 to week 15 --- p.200 / "Figure 5.9 Expression correlation between ALDH1A1 and miR-124a, -9 and -9*" --- p.201 / Figure 5.10 Expression signals of REST and SCP1 in fibroblast，fetal retina and retinoblastoma tissues --- p.202 / Table 5.1 Congenital diseases from RetNet and the corresponding gene expression signal on our data --- p.203 / Table 5.2 Expression correlation coefficient of miR-124a and mRNAs from our expression data --- p.206 / Appendices / Appendix I - Category of Retinal Disease and their Related Gene and Locus (RetNet:http://www.sph.uth.tmc.edu/RetNet/) Last updated June 13，2008 --- p.207 / Appendix II - List of Taqman probes designed for real-time PCR with gene name and the corresponding annotations and sequence --- p.210 / "Appendix III - Functional annotated of highly expressed genes (signal > 100,000) from the KEGG pathways" --- p.211 / Appendix IV - LOWESS normalized expression signal with corresponding annotation and chromosome location --- p.217 / Appendix V - Various gene lists or probes lists used in the data analysis --- p.218 / References --- p.719
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_326448 |
| Date | January 2008 |
| Contributors | Wong, Hoi Kin., Chinese University of Hong Kong Graduate School. Division of Obstetrics and Gynaecology. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, xxi, 235 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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