No description available.

Both microarray and RNA-seq technologies are powerful tools which are commonly used in differential expression (DE) analysis. Gene expression levels are compared across treatment groups to determine which genes are differentially expressed. With both technologies, filtering and normalization are important steps in data analysis. In this thesis, real datasets are used to compare current analysis methods of two-color microarray and RNA-seq experiments. A variety of filtering, normalization and statistical approaches are evaluated. The results of this study show that although there is still no widely accepted method for the analysis of these types of experiments, the method chosen can largely impact the number of genes that are declared to be differentially expressed.

No description available.

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

Wong Wai Yin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references. / Abstracts in English and Chinese. / Acknowledgement --- p.i / Abstract --- p.iii / Abbreviation --- p.vii / Chapter CHAPTER 1 --- INTRODUCTION --- p.1-1 / Chapter 1.1 --- Classification of common epithelial ovarian tumors --- p.1-2 / Chapter 1.1.1 --- Serous tumors --- p.1-4 / Chapter 1.1.2 --- Mucinous tumors --- p.1-5 / Chapter 1.1.3 --- Endometrioid tumors --- p.1-6 / Chapter 1.1.4 --- Clear cell tumors --- p.1-6 / Chapter 1.1.5 --- Cancer staging --- p.1-7 / Chapter 1.1.6 --- Tumor grading --- p.1-8 / Chapter 1.2 --- Etiology --- p.1-10 / Chapter 1.2.1 --- Factors associated with increased risks --- p.1-10 / Chapter 1.2.2 --- Factors associated with decreased risks --- p.1-12 / Chapter 1.2.3 --- Other factors --- p.1-13 / Chapter 1.3 --- Understanding of progression of ovarian carcinoma --- p.1-13 / Chapter 1.4 --- Current screening test for ovarian cancer --- p.1-15 / Chapter 1.4.1 --- Transvaginal utrasound --- p.1-15 / Chapter 1.4.2 --- Serum tumor markers --- p.1-16 / Chapter 1.5 --- Molecular basis of ovarian cancer --- p.1-18 / Chapter 1.5.1 --- Loss of heterozygosity --- p.1-18 / Chapter 1.5.2 --- Microsatellite instability --- p.1-19 / Chapter 1.5.3 --- Oncogenes --- p.1-19 / Chapter 1.5.4 --- Tumor suppressor genes --- p.1-21 / Chapter 1.6 --- Microarray gene expression profiling analysis --- p.1-25 / Chapter 1.6.1 --- Princeple of DNA micorarray --- p.1-26 / Chapter 1.6.2 --- Types of microarray --- p.1-29 / Chapter 1.7 --- Gene expression profiling of ovarian cancer --- p.1-29 / Chapter 1.7.1 --- Up-regulated genes in ovarian cancer --- p.1-30 / Chapter 1.7.2 --- Down-regulated genes in ovarian cancer --- p.1-32 / Chapter 1.8 --- Project aims --- p.1-35 / Chapter CHPATER 2 --- MATERIALS AND METHODS --- p.2-1 / Chapter 2.1 --- Materials --- p.2-1 / Chapter 2.1.1 --- Patients --- p.2-1 / Chapter 2.1.2 --- Ovarian tissue specimen --- p.2-1 / Chapter 2.2 --- Methods --- p.2-2 / Chapter 2.2.1 --- Preparation of OCT-embedded Specimen Sections --- p.2-2 / Chapter 2.2.2 --- Microdissection of Tumor Cells from Specimen Sections --- p.2-3 / Chapter 2.2.3 --- Disruption of normal ovarian frozen tissue --- p.2-3 / Chapter 2.2.4 --- Total RNA Extraction --- p.2-3 / Chapter 2.2.4.1 --- RNA Isolation --- p.2-4 / Chapter 2.2.4.2 --- DNase I Digestion --- p.2-4 / Chapter 2.2.4.3 --- RNA Cleanup and Elution --- p.2-5 / Chapter 2.2.5 --- Oligonucleotide Microarray --- p.2-6 / Chapter 2.2.5.1 --- Two-Cycle cDNA Synthesis --- p.2-6 / Chapter 2.2.5.2 --- Synthesis of Biotin-Labeled cRNA --- p.2-9 / Chapter 2.2.5.3 --- Fragmenting the cRNA for Target Preparation --- p.2-9 / Chapter 2.2.5.4 --- Target Hybridization --- p.2-10 / Chapter 2.2.5.5 --- "Array Washing, Staining, and Scanning" --- p.2-11 / Chapter 2.2.5.6 --- Statistical Analysis of Microarray Data --- p.2-11 / Chapter 2.2.6 --- Quantitative Real-time Polymerase Chain Reaction --- p.2-13 / Chapter 2.2.6.1 --- Primer and Probe --- p.2-13 / Chapter 2.2.6.2 --- Reverse-transcription --- p.2-13 / Chapter 2.2.6.3 --- Plate Setup --- p.2-14 / Chapter 2.2.6.4 --- Fluocogenic PCR --- p.2-14 / Chapter 2.2.6.5 --- Statistical Analysis of Quantitative Real-time PCR Data --- p.2-15 / Chapter CHAPTER 3 --- RESULTS --- p.3-1 / Chapter 3.1 --- Microarray gene expression data analysis --- p.3-1 / Chapter 3.1.1 --- Unsupervised Gene Selection --- p.3-1 / Chapter 3.1.2 --- Supervised Gene Selection --- p.3-3 / Chapter 3.1.2.1 --- Gene expression profiles distinguish Serous Epithelial Ovarian Tumor from Normal Ovary and identifydifferentially expressed genes --- p.3-3 / Chapter 3.1.2.2 --- Gene expression profiles distinguish Advanced Stage Serous Epithelial Ovarian Tumor from Early Stage Serous Epithelial Ovarian Tumor and identify differentially expressed genes --- p.3-22 / Chapter 3.1.2.3 --- Gene expression profiles distinguish Metastatic Serous Epithelial Ovarian Tumor from Primary Serous Epithelial Ovarian Tumor and identify differentially expressed genes --- p.3-24 / Chapter 3.2 --- Validation of microarray data by quantitative Real-time PCR --- p.3-27 / Chapter 3.2.1 --- Fold change of candidate genes --- p.3-27 / Chapter 3.2.2 --- Correlation between microarray and quantitative real-time PCR results --- p.3-29 / Chapter 3.2.3 --- Comparison of the expression of candidates genes among the different histological types of epithelial ovarian tumors --- p.3-32 / Chapter CHAPTER 4 --- DISCUSSION --- p.4-1 / Chapter 4.1 --- Global gene expression profiling using oligonucleotide microarray --- p.4-1 / Chapter 4.1.1 --- "Sensitivity, specificity and reproducibility of the Affymetrix GeneChip® microarray" --- p.4-1 / Chapter 4.1.2 --- Microarray analysis software --- p.4-3 / Chapter 4.1.2.1 --- DNA-Chip Analyzer software --- p.4-3 / Chapter 4.1.2.2 --- Comparison of statistical methods for analysis of Affymetrix GeneChip® microarray data --- p.4-5 / Chapter 4.2 --- Validation of microarray data --- p.4-7 / Chapter 4.2.1 --- Advantages of using real-time PCR for mRNA quantification --- p.4-8 / Chapter 4.2.2 --- Comparison of mRNA gene expression by RT-PCR and DNA microarray --- p.4-9 / Chapter 4.3 --- Gene expression profiling in serous ovarian cancer compared with normal ovarian epithelium --- p.4-10 / Chapter 4.3.1 --- Potential biomarkers or therapeutic targets in ovarian cancer --- p.4-12 / Chapter 4.4 --- Gene expression profiling in advanced serous ovarian cancer compared with early ovarian cancer --- p.4-16 / Chapter 4.4.1 --- Potential prognostic markers or therapeutic targets in advanced ovarian cancer --- p.4-17 / Chapter 4.5 --- Gene expression profiling in metastatic cancer compared with primary ovarian cancer --- p.4-22 / Chapter 4.5.1 --- Potential predictive markers or therapeutic targets in metastatic cancer of ovary origin --- p.4-23 / Chapter CHAPTER 5 --- CONCLUSIONS --- p.5-1 / Chapter CHAPTER 6 --- FUTURE PROSPECT --- p.6-1 / REFERENCES --- p.R-1

by Hinny Shuk-Yee Lam. / Year shown on spine: 1997. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 92-117). / Declaration --- p.i / Acknowledgements --- p.ii / Abstract --- p.iii / Table of Contents --- p.v / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Pancreas and Islets of Langerhans --- p.1 / Chapter 1.1.1 --- Islet Hormones and Glucose Balance --- p.3 / Chapter 1.1.2 --- Glucagon and Its Derived Peptides --- p.4 / Chapter A. --- Tissue-specific Post-translational Processing --- p.4 / Chapter B. --- Features of Proglucagon Gene --- p.6 / Chapter 1.1.3 --- Insulin and Features of Its Gene --- p.9 / Chapter 1.2 --- Regulation of Islet Hormone Secretion --- p.12 / Chapter 1.2.1 --- Endocrine Control --- p.12 / Chapter A --- GIP --- p.13 / Chapter B. --- Truncated GLP-1 --- p.13 / Chapter 1.2.2 --- Paracrine Control --- p.14 / Chapter 1.2.3 --- Neuroendocrine Control --- p.15 / Chapter 1.3 --- Neuropeptide Y --- p.16 / Chapter 1.3.1 --- NPY in Central Nervous System --- p.17 / Chapter 1.3.2 --- NPY in Pancreas --- p.17 / Chapter 1.3.3 --- NPY and Islet Hormones --- p.18 / Chapter 1.4 --- Synthesis and Secretion --- p.19 / Chapter 1.5 --- Objectives of Study --- p.23 / Chapter Chapter 2 --- Materials and Methods --- p.26 / Chapter 2.1 --- Effects of NPY on Islet Gene Expression --- p.26 / Chapter 2.1.1 --- Tissue Culture --- p.26 / Chapter A. --- Materials --- p.26 / Chapter B. --- Maintenance and Passage --- p.26 / Chapter C. --- Experimental Protocol --- p.28 / Chapter 2.1.2 --- Total RNA Isolation --- p.28 / Chapter A. --- Materials --- p.28 / Chapter B. --- Extraction Using FastPrep System --- p.29 / Chapter C. --- Quantification of RNA --- p.30 / Chapter D. --- Preparation of Reagents --- p.30 / Chapter 2.1.3 --- Northern Blot Analysis --- p.31 / Chapter A. --- Materials --- p.31 / Chapter B. --- Formaldehyde Gel Electrophoresis --- p.32 / Chapter C. --- Transfer onto Nylon Membrane --- p.33 / Chapter D. --- Labeling of cDNA Probes --- p.34 / Chapter E. --- Hybridization and Autoradiography --- p.35 / Chapter F. --- Preparation of Reagents --- p.36 / Chapter 2.1.4 --- Preparation of cDNA Probe --- p.37 / Chapter A. --- Materials --- p.37 / Chapter B. --- Preparation of Competent Cells --- p.37 / Chapter C. --- Transformation --- p.38 / Chapter D. --- Plasmid DNA Isolation --- p.39 / Chapter E. --- Restriction Enzyme Digestion --- p.41 / Chapter F. --- Agarose Gel Electrophoresis --- p.42 / Chapter G. --- Isolation of DNA Fragments --- p.42 / Chapter H. --- Preparation of Reagents --- p.43 / Chapter 2.1.5 --- Data Analysis --- p.46 / Chapter 2.2 --- Effects of NPY on Cytosolic Calcium --- p.46 / Chapter 2.2.1 --- Tissue Culture --- p.47 / Chapter 2.2.2 --- Confocal Laser Scanning Microscopy --- p.47 / Chapter A. --- Materials --- p.47 / Chapter B. --- Loading of Dye --- p.48 / Chapter C. --- Cytosolic Calcium Measurement --- p.49 / Chapter D. --- Preparation of Reagents --- p.49 / Chapter Chapter 3 --- Results --- p.51 / Chapter 3.1 --- Studies on Islet Gene Expression --- p.51 / Chapter 3.1.1 --- Effect of NPY on Proglucagon Expression --- p.51 / Chapter A. --- Effect at 11 mM Glucose --- p.51 / Chapter B. --- Effect at 5 mM Glucose --- p.52 / Chapter 3.1.2 --- Effect of NPY on Proinsulin Expression --- p.52 / Chapter 3.1.3 --- "Effect of PYY, PP and FSK on Proglucagon Expression" --- p.53 / Chapter 3.2 --- Studies on Cytosolic Calcium --- p.65 / Chapter 3.2.1 --- Features of InRlG9 Cells --- p.65 / Chapter 3.2.2 --- Effect of NPY on Cellular Calcium Level --- p.66 / Chapter Chapter 4 --- Discussion --- p.77 / Chapter Chapter 5 --- References --- p.92

Thesis (Ph. D.)--University of Missouri--Columbia, 2001. / Typescript. Includes bibliographical references (leaves 109-111). Also available on the Internet.

Thesis (M.S.)--University of Michigan, 2004. / Includes bibliographical references.

Thesis (Ph. D.)--University of Michigan, 2005. / Includes bibliographical references.

Thesis (Ph. D.)--University of Michigan, 2005. / Includes bibliographical references.