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Využití různých typů povrchově modifikovaných magnetických nanočástic pro izolaci a purifikaci bakteriální DNA/RNA
Bednářová, Eliška
In the submitted thesis "The use of different types of surface modified magnetic nanoparticles for isolation and purification of bacterial DNA/RNA", magnetic nanoparticles modified with tetraethoxysilane (TEOS) and (3-aminopropyl)triethoxysilane (APTES) were used. The aim was to optimize the nucleic acid isolation protocol using magnetic particles from both gram negative and gram positive bacteria in order to achieve the highest possible RNA yield. In the theoretical part of the thesis, the structure and composition of bacterial cells were summarized, especially the composition of the cell wall, which allows the bacteria to be divided into gram positive and gram negative. Furthermore, the different options for bacterial detection used in clinical practice were presented. Nucleic acid isolation is often an essential step in the detection of bacteria using molecular methods. A method based on magnetic particles is one of them, and the principle was explained here. The composition and functionalisation of the particles used for nucleic acid isolation have been described. In the practical part of the thesis, magnetic particles were characterized by several methods to specify their properties. Thus, the most suitable particles for nucleic acid isolations was selected. Next, the particles were used to optimize the procedure for nucleic acid isolation from cell lysate. The lysis buffer with 30% Triton X-100 detergent was determined to be the most effective and similar results were obtained with the 30% EcoSurf detergent. Subsequently, a method for efficient bacterial cell lysis was optimalized. Several methods were used and the best results were achieved by mechanical homogenization with 0.1 mm zirconia beads in combination with lysis buffer with 1% EcoSurf detergent. RNA yields from isolations by magnetic particles were compared with those from column isolations using a commercial isolation kit. Approximately 350 ng/µl of RNA yield was achieved for S. aureus using the column method, while 70 ng/µl less was achieved using magnetic particles. RNA yields were significantly higher than those using custom buffers, therefore, further optimization of the isolation protocol and composition of custom buffers in nucleic acid isolation would be advisable. Magnetic particles with both TEOS and APTES modifications have great potential for use in the isolation of nucleic acids from bacteria.
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Laboratory diagnostics of micrometastases in breast cancer patients
Mikulová, Veronika ; Zima, Tomáš (advisor) ; Svoboda, Marek (referee) ; Průša, Richard (referee)
Introduction: The presence of circulating tumor cells (CTC) in the peripheral blood has been associated with worse prognosis and early relapse in breast cancer patients. CTC determination in the peripheral blood has been considered as a liquid biopsy. The aim of this project was to analyze the presence of CTC followed by their molecular characterization with the potential use not only as a new biomarker for real-time monitoring of therapy efficacy but also as a suitable tool for patient's stratification and individualization of treatment for breast cancer. Methods: A total of 54 patients with diagnosed early breast cancer were enrolled into a prospective study. Ten millilitres of peripheral blood were sequentially collected to test for the presence and characterization of CTC during the follow-up of patients. CTC isolation and detection was performed by AdnaTest BreastCancer™ (AdnaGen AG, Germany), which is based on the detection of EpCAM, HER2 and MUC1 specific transcripts in enriched CTC- lysates. cDNA from isolated CTC has been further used for newly optimized qPCR assays for breast tumor and therapy resistance associated genes: TOP1, TOP2A, CSTD, ST6GAL, KRT19 and reference gene actin. qPCR results have been analyzed by Genex software (MultiD Analysis). Results: 195 blood samples have been...
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