DETECTION OF PROSTATE SPECIFIC mRNAs IN CIRCULATION AND IN PROSTATIC TISSUE

.........................................................................................7 1. REVIEW OF THE LITERATURE.............................................................9 1.1. Prostate cancer...................................................................................9 1.2. Prostate specific antigen (PSA) and human glandular kallikrein (hK2)................11 1.2.1. Genes, mRNAs and proteins.........................................................11 1.2.2. Expression and function...............................................................12 1.2.3. Clinical aspects........................................................................ 13 1.3. Prostate specific membrane antigen (PSM)................................................14 1.3.1. Gene, mRNA and protein.............................................................14 1.3.2. Expression and function.............................................................. 15 1.3.3. Clinical aspects........................................................................ 16 1.4. Genetic changes in prostate cancer..........................................................16 1.4.1. Germ line changes.....................................................................16 1.4.2. Somatic changes........................................................................17 1.5. RT-PCR techniques............................................................................19 1.5.1. RNA isolation...........................................................................19 1.5.2. Reverse transcription.................................................................. 20 1.5.3. Polymerase chain reaction............................................................21 1.5.4. Detection and quantification of the RT-PCR products...........................21 1.5.4.1. Real time quantification................................................21 1.5.4.2. Endpoint quantification.................................................22 1.5.5. Internal standards and normalization................................................24 1.6. Hematogenous spread of prostate cancer...................................................26 2. AIMS OF THE STUDY........................................................................ 30 3. MATERIALS AND METHODS.............................................................. 31 3.1. Samples..........................................................................................31 3.1.1. Cultured cells........................................................................... 31 3.1.2. Blood samples......................................................................... 31 3.1.3. Tissue samples......................................................................... 31 3.2. Detection and quantification of the mRNA................................................ 31 3.2.1. RNA extraction........................................................................ 31 3.2.2. Determination of the detection limit................................................32 3.2.3. Oligonucleotide primers.............................................................. 32 3.2.4. Internal standards......................................................................32 3.2.5. RT-PCR................................................................................. 33 3.2.5.1. Qualitative assays....................................................... 33 3.2.5.2. Quantitative assays......................................................33 3.2.6. Restriction enzyme analysis, cloning and sequencing of the PCR products...33 3.2.7. Southern blot analysis.................................................................34 3.2.8. Solid-phase minisequencing......................................................... 34