Expression profiling using manifold supports

Sammanfattning: Analyses of mRNA provides a condensed view of gene structure and quantitative analysis can reveal the induction of physiological or pathological gene expression programs. This thesis describes a new method for mRNA isolation, followed by sensitive real time detection via polymerase chain reaction (PCR), in order to quantitate transcripts of interest.Chimeric genes that result from chromosomal translocations can be used as disease-specific markers for the malignant clone to detect minimal residual diseases. It is important to detect an expanding clone as early as possible to increase the chance of a successful treatment. Accordingly, RT-PCR (reverse transcription PCR) of such chimeric transcripts has gained interest as a means to monitor patients due to its sensitivity. Expression of BCR-ABL in bone marrow or blood can be used as a measure of minimal residual disease (MRD) in patients with chronic myeloid leukemia. The newly described method for mRNA isolation was used to analyse the tumor burden in patient samples via real time detection using PCR. The proposed method constitues a promising, reproducible, and sensitive means to quantify BCR-ABL mRNA and it is suitable to monitor MRD in leukemic patients.Recombinant human erythropoietin (r-HuEpo) has an important role in the treatment of anemic patients. ?-globin mRNA was monitored in order to elucidate if it could serve as a new marker for monitoring the response to r-HuEpo. Because of the high cost of EPO treatment, an early indicator of whether a patient responds to the therapy would be of great value. The response pattern for mRNA was compared to the reticulocyte count, levels of hemoglobin, transferrin receptor and ferritin in healty individuals receiving r-HuEpo or in controls. Following treatment, ?-globin mRNA showed a more distinct increase compared to all other laboratory measurements and is therefore promising as a marker for the response to EPO treatment.The fourth project was undertaken to investigate fluctuations of mRNA expression levels for cytokines important for the rejection of xenotransplants. Porcine islet xenotransplantation could potentially solve the problem of the limited supply of suitable human donors for transplanation of islets, in order to offer a curative treatment of insulin dependent diabetes mellitus (IDDM). The rejection process was studied in the pig?rat model. Earlier studies reported a Th2 associated response. However, both morphological pattern and mRNA expression profiling supported the view that rejection is primarily due to a Th1 response.