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TaqMan-based nucleic acid quantification - abilities and limits with regards to type of collection, age and quality of human specimen
Herzogová, Eva ; Daňková, Pavlína (advisor) ; Kološtová, Katarína (referee)
Real-time PCR method is a type of PCR which allows continual monitoring of DNA amplification during every cycle of its process. It is mostly used for gene expression analysis. Based on the results of previous experiments, we decided to test out the effect of anticoagulants EDTA, heparin, sodium citrate and CPDA on the expression of selected genes of the immunological spectrum and further, to test how the time period between drawing the blood and processing of blood sample influences mRNA levels of selected genes that are determined by changes in gene expression and/or mRNA degradation. To quantify mRNA of the studied genes, we isolated total RNA from the peripheral blood leucocytes and transcribed it into cDNA by using the reverse transcription PCR. This cDNA served as a template for the real-time PCR. To examine the changes of the expression caused by the effect of each particular anticoagulants, peripheral blood derived from 10 volunteers was used (each donor's blood was taken into 3 vacuum tubes with EDTA, heparin and sodium citrate anticoagulant agents). Next to that, we obtained 10 buffy coat samples in transfusion blood bags with CPDA anticoagulant agent. Compared to blood cells influenced by one of the three anticoagulant agents present in vacuum tubes, cells from transfusion bags affected...
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Mitogenomic phylogeography and adaptive evolution of the bank vole Clethrionomys glareolus
Filipi, Karolína ; Kotlík, Petr (advisor) ; Munclinger, Pavel (referee)
This thesis is a part of the project aimed at sequencing the genome and transcriptome of the bank vole (Clethrionomys glareolus). The role of natural selection in the evolution of mitochondrial DNA (mtDNA) has been subject to much discussion; while some studies did not provide evidence that selection affected the phylogeography of the studied species, other considered adaptive evolution important. The bank vole is the key model we use to study the adaptation to climate change. As with other species, the phylogeography of the bank vole has been based on the variation of a small part of mtDNA. The goal of the thesis was to sequence the entire mitochondrial genome for representatives of all main mtDNA lineages of the bank vole using the Sanger and Illumina technologies, and to assess the role of selection and adaptation in the evolution and phylogeography of this species. The adaptive evolution in mtDNA probably was not the main driving force during the postlacial colonization of Europe. However, signatures of adaptive evolution have been found - an amino acid change with possible functional consequences in one gene and an excess of radical changes in physical- chemical properties of amino acids in populations at the latitudinal (northern and southern) extremes of the bank vole distribution. Key...
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Klastrování genů s podobným regulačním mechanismem během monocytární a garanulocytární diferenciace buněk HL-60
Koutná, I. ; Faltýsková, E. ; Krontorád, P. ; Svoboda, Z. ; Pavlík, T. ; Bártová, Eva ; Kozubek, Michal ; Kozubek, Stanislav
In the present study, a high density-microarrays of human cDNA containing 19,000 genetic elements were used to search for differences in gene expression of human promyelic leukaemia cell line HL-60 during monocyte (MD) and granulocyte differentiation (GD). For differentiation to monocytes (granulocytes), the TPA, 12-O-tetradecanoylphorbol-D-acetate (DMSO) were used, respectively. A total number of 4625 (4760) genes were found to be regulated during MD (GD). The genes have been divided into groups according to their kinetics of regulation (R-groups). The genes of the same group are not distributed randomly throughout the genome but form clusters on chromosomes (R-clusters). Thus, functionally interconnected genes are frequently localized near to each other on the DNA molecule.
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