- Towards molecular medicine:optimization of the methods for gene expression analysis in clinical samples.
- Dotti, Isabella
Subject
- molecular medicine, gene expression analysis, clinical samples, technical optimization
- MEDICINA MOLECOLARE
- BIO/11 BIOLOGIA MOLECOLARE
Description
- 2006/2007
- The advent of molecular “-omics” technologies enabled an unprecedented view into the inner molecular mechanisms of cancer and enhanced optimism towards a patient-tailored vision of medicine. The successful application of these molecular approaches in the discovery of candidate biomarker has accelerated the shift towards personalization of medicine. Indeed, biomarkers hold great promise for refining our ability to establish early diagnosis and prognosis, and to predict response to therapy. The develoment of clinically useful biomarkers would be impossible without access to human biological specimens and associated patient data, since they complete the molecular information gained from laboratory research. Furthermore, with the advances of sensitive molecular technologies, human bio-specimens can be now successfully used for wide analysis at all molecular levels (DNA, RNA and proteins), in addition to conventional cytologic and histologic investigations. However, despite the hundreds of reports on tumor markers, only a few markers have proven clinically useful. The insufficient experience in clinical application of molecular methods combined with the high complexity of clinical material represent the major obstacles for the development of clinically useful biomarkers. Thanks to the possibility to have access to the fresh and archival samples from the hospital, our laboratory can investigate the potential of technological innovations and the current technical pitfalls directly on clinical material. The work in my thesis is strictly correlated to this activity. In particular, the first part is focused on the technical optimization of molecular methods for gene expression analysis in biological fluids and especially in urine samples. In this context we validated a new experimental kit for total RNA extraction from urine samples and tested the potential of a colorimetric approach for PCR product detection. The major part of the study is focused on the technical optimization of molecular methods for gene expression analysis in archival material. This activity is in step with one of the main objectives of the European project called “Archive tissues: improving molecular medicine research and clinical practice-IMPACTS”, in which my laboratory and other 20 European centres are directly involved. In this phase the comparison of the experiences between laboratories and their active collaboration are essential for a more rapid validation of protocols dedicated to RNA (but also DNA and protein) analysis. In particular, we investigated some molecular aspects involved in the pre-analytical phase (tissue fixation procedures) and analytical phase (RNA extraction, RNA quantification and integrity assessment, qRT-PCR) of tissue processing. The final objective of this activity will be the definition of common technical guidelines for a reliable quantification of molecular biomarkers for diagnosis, prognosis and therapy directly in human archival samples. Finally, my thesis includes the clinical application of molecular methods for the quantification of candidate biomarkers in two archival case studies (a breast cancer and an adrenal gland cancer case study). In the breast cancer case study we showed that a panel of seven genes (involved in different cell pathways) is associated to patients’ survival. The adrenal gland tumor case study is part of a preliminary study about the angiogenetic process in rare human cancers.
- 1979
Date
- 2008-05-07T12:07:24Z
- 2008-05-07T12:07:24Z
- 2008-04-01
Type
- Doctoral Thesis
Format
- application/pdf
Identifier
urn:nbn:it:units-7204