Expression analysis of a selected gene set in malignant and non-malignant tissues derived from individuals with colon cancer [Elektronische Ressource] : comparison witz protein expression data / von Maria Radeva

Mradeva

Sujets

• Gesundheit

Informations

• Publié par: friedrich-schiller-universitat_jena
• Publié le: 01 janvier 2009
• Nombre de lectures: 39
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

Expression analysis of a selected gene set in malignant
and non-malignant tissues derived from individuals with
colon cancer. Comparison with protein expression data.






Dissertation
zur Erlangung des akademischen Grades
doctor rerum naturalium (Dr. rer. nat.)






vorgelegt dem Rat der Biologisch-Pharmazeutischen Fakultät
der Friedrich-Schiller-Universität Jena




von





Maria Radeva, M.Sc.
geboren am 05.02.1977 in Sofia, Bulgarien























Die vorliegende Arbeit wurde durchgeführt am:




1. Gutachter: Prof. Dr. Karl Otto Greulich
Friedrich-Schiller Universität Jena

2. Gutachter: PD. Dr. Michael Glei


3. Gutachter: PD. Dr.rer.nat. Dr.med. Zeno Földes-Papp
University of North Texas


Tag der öffentlichen Verteidigung: 20.07.2009



Dedicated to my family
























PUBLICATIONS

Parts of this work are included in the following publications:

Radeva M, Hofmann T, Altenberg B, Mothes H, Richter KK, Pool-Zobel B, Greulich KO
(2008):
The database dbEST correctly predicts gene expression in colon cancer patients. Curr Pharm
Biotechnol.;9(6):510-5

Radeva M, Jahns F, Wilhelm A, Glei M, Settmacher U, Greulich KO, Mothes H: A burst of
alpha-defensin 6 expression in the adenoma stage of human colon carcinogenesis, in
preparation
TABLE OF CONTENTS
TABLE OF CONTENTS


TABLE OF CONTENTS ..................................................................................................................................I
ACKNOWLEDGMENTS.............................................................................................................................. IV
ABBREVIATIONS.........................................................................................................................................VI
SUMMARY................................................................................................................................................... VII
ZUSAMMENFASSUNG.............VIII
1. INTRODUCTION................................................................................................................................... 1
1.1. DIAGNOSTIC APPROACHES TO PROVE THE DETECTION OR TREATMENT OF COLORECTAL CANCER
(CRC) ......................................................................................................................................................... 1
Access to tissues of different stages of colon carcinogenesis........................................................................... 2
1.2. COLORECTAL CANCER DEVELOPMENT ............................................................................................. 3
1.3. WNT SIGNALLING PATHWAY ............................................................................................................ 5
1.4. BASIC KNOWLEDGE ABOUT THE GENES UNDER INVESTIGATION AND THE ENCODING BY THEM
PROTEINS........................................................................................................................................................ 7
Osteopontin (OPN)........................................................................................................................................... 7
Cyclooxygenase-2 (COX-2)............................................................................................................................. 7
DEAD (Asp-Glu-Ala-Asp) box polypeptide 6 (DDX6)................................................................................... 8
Eukaryotic initiation factor 4E (eIF4E)............................................................................................................ 9
Histone Acetyl Transferase 1 (HAT1).............................................................................................................. 9
Ubiquitin Specific Protease 28 (USP28) ........................................................................................................ 10
Polo-like kinase 1 (PLK1).............................................................................................................................. 10
Pyruvate kinase type M2 (PKM2).................................................................................................................. 11
Heat Shock Proteins 90 beta (HSP90ß).......................................................................................................... 12
Defensin alpha (DEFA 1-3 and DEFA 6)....................................................................................................... 12
2. AIMS OF THE WORK ........................................................................................................................ 14
3. MATERIALS AND METHODS ......................................................................................................... 15
3.1. MATERIALS.................................................................................................................................... 15
3.1.1. Chemicals and Kits................................................................................................................... 15
3.1.2. Cell lines for gene expression analysis..................................................................................... 16
3.1.3. Medium and reagents for cultivation of cell lines..................................................................... 16
3.1.4. Antibodies and recombinant proteins ....................................................................................... 17
3.2. METHODS........ 17
3.2.1. Tissue sample preparation for gene and protein expression analysis ...................................... 17
3.2.2. Agarose gel electrophoresis...................................................................................................... 19
Ethidium bromine gel electrophoresis............................................................................................................ 19
Formaldehyde agarose gel electrophoresis.............. 19
3.2.3. Gene expression analysis.......................................................................................................... 19
Total RNA extraction from tissue samples and cell lines............................................................................... 19
RNA integrity................................................................................................................................................. 20
Complementary DNA (cDNA) synthesis ....................................................................................................... 20
Genomic DNA (gDNA) extraction ................................................................................................................ 20
Design of primers........................................................................................................................................... 20
Verification of the primer specificity ............................................................................................................. 21
Quantitative Real - Time PCR (qRT-PCR) conditions................................................................................... 23
PCR reaction efficiency ................................................................................................................................. 23
3.2.4. Protein expression analysis. ..................................................................................................... 23
I

TABLE OF CONTENTS
3.2.4.1. Protein extraction ........................................................................................................................... 23
Preparation of lysates from tissue samples..................................................................................................... 23
3.2.4.2. SDS PAGE and Western blot, quantification by ECL.................................................................... 24
3.2.4.3. Quantification of the chemiluminescent signal by densitometry and evaluation of the Western blot
data ........................................................................................................................................................ 25
3.2.5. Data mining, evaluation techniques and statistical methods.................................................... 25
3.2.5.1. Data mining.................................................................................................................................... 25
3.2.5.2. Principal Component Analysis (PCA)-the idea behind it ............................................................... 25
Background of PCA. Description and terminology........................................................................................ 28
Description of the software used for PCA analysis ........................................................................................ 30
3.2.5.3. Relative expression software tool (REST)..........