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Gene expression analysis in non small cell lung cancer (NSCLC) using microarray technology
S116
Abstracts
4.05 #141
ADJUSTING THE AMPLICON SIZE IN DOP-PCR V.J. Leon, T. Talaban. Servicio de Bioquimica, Hospital Universitario de Salamanca, Salamanca, Spain With only small quantities of DNA, the use of DOP-PCR greatly increases the quantity of genomic material available by employing primer strands which join up with fixed segments of the DNA chain. We have found that the application of this technique for conditions and components that exist in the kinetics commonly described in the literature leads to an extremely high replication of amplifications of approximately 500 bp, in comparison to larger amplifications, which means that when we work with DNA segments of over 800 bp we have to increase the quantity of the amplified target in order to obtain the desired results. The analysis of the chemical components of the reaction mixture reveals its kinetic role: the salts’ lends the necessary osmotic strength to allow for the correct hybridisation of the DNA primer chain and together with the buffer, creates the optimum conditions for the ploymerase Taq enzyme to work. The concentration of nucleotides and magnesium maximises their access to the enzyme. The programming of the physical conditions of the thermocycler ensures that the starting blocks and the optimum temperature necessary for DOP-PCR kinetics are obtained. Material and Methods: Series of reaction mixtures were set up in which from 100 ng of DNA, in each reaction, the concentrations of primers, magnesium, NTPs, and reaction pH values were varied. The physical conditions of the thermocycler were standard, as recommended by the Roche DOP-PCR kit, which we used as a parallel control to our own programmed series. Results: The analysis of the series of chemical conditions studies revealed that the median size of the amplifications obtained is proportionate to the salt concentration, thereby creating a tool which will allow us to widen the applications of DOP-PCR.
4.05 #142
GENE EXPRESSION ANALYSIS IN NON SMALL CELL LUNG CANCER (NSCLC) USING MICROARRAY TECHNOLOGY Paolo Fardin,2 Jurg Bahler,3 Paolo Capanni,2 Erika Inglese,2 Annamaria Ricciardi,2 Giovanni B. Ferrara.1,2 1Immunogenetics, National Cancer Research Institute, Genova, Italy; 2Oncology, Biology and Genetics, University of Genoa, Genoa, Italy; 3the Wellcome Trust Sanger, Cambridge, United Kingdom Non small cell lung cancer (NSCLC) represents the most frequent histological subtype of all lung tumor cases. The identification of prognostic factors could be very useful to understand lung cancer biology and to develop therapeutic strategies based on new drugs against molecular targets. Using cDNA microarray, we studied normal and tumoral tissues of five different patients with non small cell lung cancer. RNA samples were extracted from frozen tissues and then labeled cDNAs were synthetized and used for hybridization. In our study we used PAN Human Cancer microarray (MWG) which contains about 2,000 human genes associated with cancer development and progression. We compared gene expression levels of tumoral sample against the normal one of each patient by the use of data mining softwares and in collaboration with the Wellcome Trust Sanger Institute. 64 genes are differentially expressed at least twofold in at least 2 out of 5 samples and 9 genes are over-expressed more than 16-fold. We are studying the correlation between the expression of these genes and the histological data characterizing the different tumor subtypes. The following studies on selected genes are important to choose potential targets for therapeutic strategies. We thank ISO, Istituto Superiore di Oncologia, for the facilities and equipment.
Abstracts
4.05 #141
ADJUSTING THE AMPLICON SIZE IN DOP-PCR V.J. Leon, T. Talaban. Servicio de Bioquimica, Hospital Universitario de Salamanca, Salamanca, Spain With only small quantities of DNA, the use of DOP-PCR greatly increases the quantity of genomic material available by employing primer strands which join up with fixed segments of the DNA chain. We have found that the application of this technique for conditions and components that exist in the kinetics commonly described in the literature leads to an extremely high replication of amplifications of approximately 500 bp, in comparison to larger amplifications, which means that when we work with DNA segments of over 800 bp we have to increase the quantity of the amplified target in order to obtain the desired results. The analysis of the chemical components of the reaction mixture reveals its kinetic role: the salts’ lends the necessary osmotic strength to allow for the correct hybridisation of the DNA primer chain and together with the buffer, creates the optimum conditions for the ploymerase Taq enzyme to work. The concentration of nucleotides and magnesium maximises their access to the enzyme. The programming of the physical conditions of the thermocycler ensures that the starting blocks and the optimum temperature necessary for DOP-PCR kinetics are obtained. Material and Methods: Series of reaction mixtures were set up in which from 100 ng of DNA, in each reaction, the concentrations of primers, magnesium, NTPs, and reaction pH values were varied. The physical conditions of the thermocycler were standard, as recommended by the Roche DOP-PCR kit, which we used as a parallel control to our own programmed series. Results: The analysis of the series of chemical conditions studies revealed that the median size of the amplifications obtained is proportionate to the salt concentration, thereby creating a tool which will allow us to widen the applications of DOP-PCR.
4.05 #142
GENE EXPRESSION ANALYSIS IN NON SMALL CELL LUNG CANCER (NSCLC) USING MICROARRAY TECHNOLOGY Paolo Fardin,2 Jurg Bahler,3 Paolo Capanni,2 Erika Inglese,2 Annamaria Ricciardi,2 Giovanni B. Ferrara.1,2 1Immunogenetics, National Cancer Research Institute, Genova, Italy; 2Oncology, Biology and Genetics, University of Genoa, Genoa, Italy; 3the Wellcome Trust Sanger, Cambridge, United Kingdom Non small cell lung cancer (NSCLC) represents the most frequent histological subtype of all lung tumor cases. The identification of prognostic factors could be very useful to understand lung cancer biology and to develop therapeutic strategies based on new drugs against molecular targets. Using cDNA microarray, we studied normal and tumoral tissues of five different patients with non small cell lung cancer. RNA samples were extracted from frozen tissues and then labeled cDNAs were synthetized and used for hybridization. In our study we used PAN Human Cancer microarray (MWG) which contains about 2,000 human genes associated with cancer development and progression. We compared gene expression levels of tumoral sample against the normal one of each patient by the use of data mining softwares and in collaboration with the Wellcome Trust Sanger Institute. 64 genes are differentially expressed at least twofold in at least 2 out of 5 samples and 9 genes are over-expressed more than 16-fold. We are studying the correlation between the expression of these genes and the histological data characterizing the different tumor subtypes. The following studies on selected genes are important to choose potential targets for therapeutic strategies. We thank ISO, Istituto Superiore di Oncologia, for the facilities and equipment.