Towards a Comprehensive Description of the Human Retinal Transcriptome: Identification and Characterization of Differentially Expressed Genes Dissertation zur Erlangung des naturwissenschaftlichen Doktorgrades der Bayerischen Julius-Maximilians-Universität Würzburg vorgelegt von Heidi Schulz aus Argentinien Würzburg 2003 Eingereicht am 10. September 2003 Bei der Fakultät für Biologie Mitglieder der Promotionskommission Vorsitzender: Prof. Dr. R. Hedrich Gutachter: Prof. Dr. Bernhard H.F. Weber Gutachter: Prof. Dr. Ricardo Benavente Tag des Promotionskolloquiums: Doktorurkunde ausgehändigt am: Erklärung gemäß §4, Absatz 3 der Promotionsordnung für die Fakultät für Biologie der Universität Würzburg: Hiermit erkläre ich, dass ich die vorliegende Dissertation selbständig durchgeführt und verfasst habe. Andere Quellen als die angegebenen Hilfsmittel und Quellen wurden nicht verwendet. Die Dissertation wurde weder in gleicher noch in ähnlicher Form in einem anderen Prüfungsverfahren vorgelegt. Es wurde zuvor kein anderer akademischer Grad erworben. Die vorliegende Arbeit wurde am Institut für Humangenetik der Universität Würzburg unter der Leitung von Prof. Bernhard H.F. Weber angefertigt. Würzburg, den 10. September 2003 ACKNOWLEDGMENTS I would like to thank Prof. Bernhard Weber for giving me the possibility to do my Ph.D. research in his group and for coaching me in the fine art of scientific investigation and writing. My gratitude also extends to Prof. Ricardo Benavente who as a member of the Faculty of Biology of the University of Würzburg accepted to be supervisor of this thesis. I would like to acknowledge Dr. Heidi Stöhr for introducing me to the world of gene identification and characterization. I would also like to express my gratitude to past and present lab members of the AG Weber who helped me and contributed to a very pleasant working atmosphere. In particular I thank Jelena Stojic for the great working relationship, Vladimir Milenkovic for his patient assistance in all computer-related problems, Susanne Fröhlich and Claudia Berger for helping with the RT-PCR expression analyses, Franziska Krämer and Christine Wiedemann for the proofreading assistance, and Andrea Rivera for always being there. I am deeply indebted to my family. You really deserve far more credit than I can ever give you! Just to mention a few things, I thank you for your generous love, support, advice, and for teaching me not with words but with your actions and life. Your integrity and warmth provided a wonderful environment in which to grow. My most heartfelt thanks go to my husband Lucio. Without his strength, patience, and comprehension, it would not have been possible for me to finish this dissertation. Through the hardships of graduate studies, my resolve remained strong because of his love and understanding. My final recognition and thanks go to God for His everlasting love, guidance, the health He gave me these years, and for being my strength. Table of Contents Table of Contents I Summary ................................................................................................................................. 1 II Zusammenfassung ........................................................................................................................... 4 III Introduction 1. The human retina ........................................................................................................................................ 7 2. Hereditary retinal degenerations ................................................................................................................. 9 2.1. The age-related macular degeneration (AMD)................................................................................... 10 3. Molecular genetics of human retinal disease ............................................................................................ 10 4. Gene identification approaches for monogenic disorders ......................................................................... 11 5. Gene identification approaches for complex disorders ............................................................................. 13 6. Contributions of the genomic era to gene identification efforts ................................................................. 15 7. Goal of the thesis ...................................................................................................................................... 18 IV Material and Methods 1. Bioinformatic tools ..................................................................................................................................... 19 1.1. Databases.......................................................................................................................................... 19 1.1.1. Sequence databases............................................................................................................... 19 1.1.1.1. GenBank ......................................................................................................................... 19 1.1.1.2. Databases of expressed sequence tags (EST)............................................................... 19 1.1.1.3. UniGene .......................................................................................................................... 19 1.1.1.4. TIGR................................................................................................................................ 20 1.1.2. Gene information databases ................................................................................................... 20 1.1.3. Databases of genes involved in disease ................................................................................. 20 1.1.4. Other databases...................................................................................................................... 21 1.1.4.1. PubMed........................................................................................................................... 21 1.1.4.2. HUGO Gene Nomenclature (HGCN) Committee............................................................ 21 1.1.4.3. HuGEIndex Gene Specific Expression database............................................................ 21 1.2. Sequence analysis tools .................................................................................................................... 21 1.2.1. Splice site discrimination score ............................................................................................... 22 2. RNA-related methods................................................................................................................................ 23 2.1. Sources.............................................................................................................................................. 23 2.1.1. Tissue ...................................................................................................................................... 23 2.1.2. RNA......................................................................................................................................... 23 2.2. RNA isolation ..................................................................................................................................... 23 2.3. Elimination of contaminating genomic DNA....................................................................................... 23 2.4. Northern blot analysis ........................................................................................................................ 24 2.4.1. Filter preparation ..................................................................................................................... 24 2.4.2. Probe labeling.......................................................................................................................... 24 2.4.3. Membrane hybridization and washing ..................................................................................... 24 2.5. First-strand cDNA synthesis............................................................................................................... 25 3. DNA-related methods................................................................................................................................ 25 3.1. Isolation of plasmid DNA.................................................................................................................... 25 3.2. Polymerase chain reaction (PCR)...................................................................................................... 25 3.3. Relative quantitative real-time PCR (qRT-PCR) ................................................................................ 26 3.3.1. Primer design .......................................................................................................................... 26 3.3.2. Optimization of qRT-PCR reactions ........................................................................................ 26 3.3.3. Determination of amplification efficiency ................................................................................. 26 3.3.4. qRT-PCR Protocol................................................................................................................... 27 3.3.5. Data analysis........................................................................................................................... 27 3.4. Agarose gel electrophoresis..............................................................................................................