Isolation and functional analysis of differentially expressed genes in human prostate cancer

Abstract

1.1 Human prostate cancer : an overview 1.2 Staging of prostate cancer 1.3 Susceptibility genes in prostate carcinoma 1.4 Diagnostic markers of prostate cancer 1.4.1 New putative prostate cancer markers identified by microarray analysis 1.4.2 Serum markers in prostate carcinognesis 1.5 Aims of the study MATERIALS AND METHODS2.1 Chemicals and reagents 2.2 Solutions and buffers 2.3 Sterilization of solutions and equipments 2.4 Bacterial strains and mediums 2.5 Eukaryotic cell lines 2.6 Plasmids and cDNA fragments 2.7 Synthetic oligonucleotide primers 2.8 Antibodies 2.9 Isolation of nucleic acids 2.9.1 Small-scale preparation of plasmid DNA (Mini prep) 2.9.2 Preparation of bacterial glycerol stocks 2.9.3 Large-scale preparation of plasmid DNA (Midi prep) 2.9.4 Isolation of total RNA from eukaryotic cells 2.9.5 Isolation of total RNA from tissues 2.9.6 Isolation of poly (A) + -enriched RNA 2.9.7 Determination of nucleic acid concentration 2.10 Cloning techniques 2.10.1 Restriction analysis of DNA 2.10.2 Isolation of DNA fragments from agarose gels 2.10.3 Dephosphorylation of 5' ends of DNA 2.10.4 Phenol-chloroform extraction and ethanol precipitation 2.10.5 Filling-up reaction of DNA ends 2.10.6 Ligation of DNA fragments 2.10.7 Subcloning of PCR and RT-PCR products 2.10.8 Transformation of competent cells with plasmid DNA 2.11 Gel electrophoresis and blotting techniques 2.11.1 Agarose gel electrophoresis of DNA 2.11.2 Agarose gel electrophoresis of RNA and Northern blot analysis Contents II 2.11.3 DNA and RNA molecular weight ladders 2.12 Sequencing 2.13 One-step RT-PCR 2.14 Labeling and hybridization of nucleic acids 2.14.1 Generation of 32 P labeled cDNA by using the "random prime" method 2.14.2 Northern blot hybridization with radioactive labeled cDNA-probes 2.15 Isolation of differentially expressed genes (Atlas Array) 2.15.1 Overview of the Atlas Array procedure 2.15.2 Preparation of whole cDNA probes from total RNA 2.15.3 DNase treatment of total RNA 2.15.4 Poly(A) + RNA enrichment 2.15.5 Whole cDNA probe synthesis 2.15.6 Purification of labeled cDNA probes by column chromatography 2.15.7 Hybridization of Atlas Arrays with labeled cDNA probes 2.16 Analysis of gene expression of multiple tumor samples (Cancer Profiling Array) 2.17 Protein techniques 2.17.1 Isolation of total proteins from eukaryotic cells 2.17.2 Determination of protein concentration 2.17.3 Electrophoresis of proteins 2.17.4 Western blotting of proteins onto nitrocellulose filters 2.17.5 Staining of polyacrylamide gel 2.17.6 Incubation of protein-bound membranes with antibodies 2.18 Preparation of laser capture microdissection-derived total RNA 2.18.1 Laser capture microdissection of prostate cancer cells 2.18.2 RNA extraction from LCM samples 2.18.3 Agilent RNA LabChip Techniques 2.19 Real-time RT-PCR analysis 2.19.1 Real time RT-PCR by using SYBR Green I dye 2.19.2 Real time RT-PCR by using standard dual-labeled probes 2.20 Eukaryotic cell culture methods 2.20.1 Cell culture conditions 2.20.2 Preparation of conditioned medium 2.20.3 Trypsiniza...