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Practical aspects of single-cell RT-qPCR analysis
Žucha, Daniel ; Valihrach, Lukáš (advisor) ; Pavlínková, Gabriela (referee)
Recent breakthroughs in the RNA quantification of single cells are rapidly transforming the view on biology and medicine. Flexibility and sensitivity of reverse transcription quantitative PCR (RT-qPCR) make it an ideal method for quantification of single-cell material, but its limits had not been yet fully explored. In this thesis, various factors influencing RT-qPCR performance in single-cell application have been assessed, including conditions of sample collection and processing, importance of quality control, performance of reverse transcription, preamplification and role of qPCR assays. We showed that prolonged time for single cell collection as well as repeated freeze-thaw cycles had negligible effect on RT-qPCR data quality. Direct lysis routinely applied for RNA extraction from single cells may be scaled up to 256 cells. The comprehensive comparison of 11 reverse transcriptases in low RNA input conditions identified 2 best-performing enzymes. Decrease in preamplification volume as well as poor primer design resulted in the loss of sensitivity. Finally, the established workflow has been applied to profile gene expression of astrocytes in mouse model of amyotrophic lateral sclerosis (ALS) identifying important components of ALS-induced changes to astrocyte transcriptome. Altogether, the thesis...
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The importance and role of reverse transcriptases in gene expression analysis
Žucha, Daniel ; Valihrach, Lukáš (advisor) ; Španielová, Hana (referee)
The continuously advancing field of gene expression analysis enables the evaluation of even the slightest changes that occur in the cell transcriptome. In order to ensure accuracy of the observed biological variances, it is fundamentally important to be aware of the possible biases introduced during sample processing. In gene expression research, the methods of reverse transcription−quantitative PCR (RT−qPCR) and RNA- Sequencing (RNA-Seq) are often the primary choice, mostly because of their high precision and reproducibility. Since these both methods require DNA template, they are coupled with the same initial step - reverse transcription (RT), a reaction producing DNA complementary to its RNA template. It is well known that RT introduces bias. As a result, it is therefore of importance to thoroughly evaluate the effects of these biases. One such annotated source of artifacts is the reverse transcriptase (RTase) itself. However, it has been shown that the enzyme does not account for most of the variance alone. Surprisingly, choice of primers or RNA template may influence the reaction outcome even more than the bias introduced from the enzyme. This is especially the case with recent advances in protein engineering. Production of highly efficient RTases may pronounce the variation originating from...
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Host factors involved in Rous Sarcoma Virus replication
Štafl, Kryštof ; Svoboda, Jan (advisor) ; Horníková, Lenka (referee)
Rous sarcoma virus (RSV) takes a place of honor among retroviruses. Research of RSV allows us to uncover the secret of the origin and evolution of life, the mechanisms of tumorigenesis and the interaction between viruses and their hosts. Viruses are not able to replicate themselves without host cells. They exploit a number of cellular pathways and factors and they can reprogram the cells to produce great amounts of viral progeny. They exert pressure on their host that leads to developement of new types of cellular proteins, which then results in resistance of the cells. This thesis focuses on the host factors involved in the RSV replication cycle. It summarizes the current knowledge about the replication cycle of retroviruses and the host factors that are necessary for productive infection: cellular receptors, endocytic and secretory pathways, nuclear transport, proteosynthesis, replication of proviruses and its stimulation. The restriction mechanisms of cells are also taken into account. The current knowledge about RSV is compared with facts on mammalian retroviruses and gaps in research are highlighted. The influence of the host cell factor absence, host specificity and cellular permissiveness are correlated and discussed.
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Detection of phytoplasmas using DNA-microarrays
MARKOVÁ, Jaroslava
The aim of this thesis was to optimize the method of detection of phytoplasmas using DNA-microarray. It consisted of testing an appropriate method of genetic material isolation, development and optimization of PCR to amplify different groups of phytoplasmas, optimization of detection of DNA at a microarray, and sequence analysis of phytoplasma in order to design more suitable probes. PCR was first optimized for collection isolates, then also for natural samples. All 16Sr groups from the collection were sequenced and phytoplasmas were detected in them by hybridization. Phytoplasmas were detected also in natural samples: oilseed rape (species Brassica napus), red clover (Trifolium pretense), purple coneflower (Echinacea purpurea), and apple tree (Malus domestica). Using the DNA from insect vectors, only sample 202/6 from the group 16Sr-XII was positive. The sequence of red clover and oilseed rape correspond with the database samples in the group 16Sr-I "Aster yellows".
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