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DNA Microarray Based Gene Expression Profiling in Human Hepatocyte Cells to Serve As a Basis for Dynamic Modelling of the Human Liver - a Systems Biology Approach

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DNA Microarray Based Gene Expression Profiling in Human Hepatocyte Cells to Serve As a Basis for Dynamic Modelling of the Human Liver - a Systems Biology Approach DNA Microarray Based Gene Expression Profiling in Human Hepatocyte Cells to Serve as a Basis for Dynamic Modelling of the Human Liver - a Systems Biology Approach Von der Fakultät Energie-, Verfahrens- und Biotechnik der Universität Stuttgart zur Erlangung der Würde eines Doktors der Naturwissenschaften (Dr. rer. nat.) genehmigte Abhandlung vorgelegt von Thomas Reichart aus Sindelfingen Hauptberichter: Prof. Dr. Rolf D. Schmid, MBA Mitberichter: Prof. Dr. Dr. h.c. Matthias Reuss Tag der mündlichen Prüfung: 10. Juni 2008 Institut für Technische Biochemie der Universität Stuttgart 2008 TABLE OF CONTENTS TABLE OF CONTENTS...........................................................................................................................2 EIDESSTATTLICHE ERKLÄRUNG .....................................................................................................5 ACKNOWLEDGMENT............................................................................................................................6 ZUSAMMENFASSUNG............................................................................................................................7 ABSTRACT ..............................................................................................................................................15 ABBREVIATIONS ..................................................................................................................................21 1 INTRODUCTION ..........................................................................................................................24 1.1 SYSTEMS BIOLOGY - A NEW APPROACH ..................................................................................24 1.2 "HEPATOSYS" - A COMPUTATIONAL APPROACH TO UNDERSTAND FUNCTIONAL ASPECTS OF THE LIVER ...........................................................................................................................31 1.3 XENOBIOTIC METABOLISM ......................................................................................................34 1.4 MICROARRAY TECHNOLOGY....................................................................................................40 1.4.1 Microarrays in Drug Discovery, Drug Development and Future Applications .................50 1.4.2 Basics of Microarray Technology ......................................................................................55 1.4.3 Data Quantification and Interpretation of Microarray Experiments .................................66 1.4.4 Identifying Differentially Expressed Genes ........................................................................69 1.5 OBJECTIVE OF THIS THESIS ......................................................................................................76 2 MATERIALS AND METHODS ...................................................................................................78 2.1 CHEMICALS, ENZYMES AND KITS ............................................................................................78 2.2 HARDWARE EQUIPMENT ..........................................................................................................79 2.3 SOFTWARE TOOLS AND INTERNET RESOURCES........................................................................80 2.4 BUFFERS AND SOLUTIONS........................................................................................................81 2.4.1 Complex media ...................................................................................................................81 2.4.2 Antibiotics...........................................................................................................................82 2.5 MEDIA FOR CULTIVATION OF PRIMARY HUMAN HEPATOCYTES ..............................................82 2.6 PRIMARY HUMAN HEPATOCYTE CELLS, BACTERIAL STRAINS AND PLASMIDS USED IN THIS WORK ..............................................................................................................................83 2.6.1 Cultivation of Bacterial Cells .............................................................................................86 2.6.2 Conservation of Strains ......................................................................................................87 2.7 CELL CULTIVATION AND INDUCTION OF PRIMARY HEPATOCYTES...........................................87 2 TABLE OF CONTENTS 3 2.7.1 Bringing Primary Hepatocytes into Culture after Transport .............................................87 2.7.2 Induction Experiments of Primary Hepatocytes.................................................................88 2.7.3 Harvesting of Primary Hepatocytes ...................................................................................89 2.8 METHODS OF MOLECULAR BIOLOGY .......................................................................................90 2.8.1 Working with RNA..............................................................................................................90 2.8.2 Purification of Nucleic Acids..............................................................................................91 2.8.3 Quality Control of Nucleic Acids........................................................................................92 2.8.4 Precipitation of Nucleic Acids............................................................................................94 2.8.5 RNA Amplification..............................................................................................................94 2.8.6 Digestion of DNA..............................................................................................................100 2.8.7 Separation of Nucleic Acids Using Agarose Gel Electrophoresis....................................101 2.8.8 Preparation and Transformation of Competent E. coli . ..................................................104 2.8.9 Reverse Transcription.......................................................................................................106 2.8.10 Quantitative Real-time PCR ........................................................................................108 2.9 PROTEIN BIOCHEMICAL METHODS.........................................................................................114 2.9.1 Protein Precipitation with Acetone...................................................................................114 2.9.2 SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)..................................................114 2.9.3 Semi-dry Protein Transfer for Western Blotting...............................................................119 2.9.4 Immunological Detection of Proteins on Nitrocellulose Filters.......................................120 2.10 METHODS USING DNA MICROARRAYS ..................................................................................122 2.10.1 Labeling of aRNA/cDNA..............................................................................................122 2.10.2 Indirect Labeling of Amplified (a)RNA........................................................................122 2.10.3 Direct Labeling During Reverse Transcription ...........................................................123 2.10.4 Indirect Labeling During Reverse Transcription.........................................................125 2.10.5 Hybridization and Washing .........................................................................................127 2.10.6 Detection of Indirectly Labeled Targets ......................................................................129 2.10.7 Scanning of Arrays.......................................................................................................129 2.10.8 Extraction of Raw Data and Picture Analysis (Quantification)...................................130 2.10.9 Evaluation of DNA Microarrays and Data Analysis ...................................................131 2.10.10 Analysing Sub-Genome Data using the Eppendorf DualChip® Software................132 2.10.11 Analysing Full-Genome Data Using "Refiner" and "Analyst" from Genedata..........138 2.10.12 Literature Mining Using Bibliosphere from Genomatix..............................................147 3 RESULTS ......................................................................................................................................152 3.1 COMPARISON OF MICROARRAYS FROM DIFFERENT SUPPLIERS..............................................153 3.2 EXPERIMENTAL SETUP OF TIME-SERIES EXPERIMENTS..........................................................162 3.3 RNA ISOLATION AND QUALITY CONTROL.............................................................................165 3.4 VERIFICATION OF INDUCTION OF CELL CULTURES.................................................................173 3.4.1 Quantitative Real-Time PCR (qRT-PCR).........................................................................173 4 Introduction 3.4.2 Western Blotting ...............................................................................................................177 3.5 REVERSE TRANSCRIPTION (RT), AMPLIFICATION AND LABELING OF MRNA.........................178 3.5.1 Direct Labeling with Fluorescent Dyes............................................................................178 3.5.2 Amplification and Indirect Labeling Using Biotin Modified Nucleotides.........................181 3.6 HYBRIDIZATION OF ARRAYS AND QUALITY CONTROL...........................................................185 3.6.1 Quality Control for Sub-Genome Arrays from Eppendorf................................................185
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