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Influence of carrier molecules addition on plasma free circulating nucleic acids extraction
Vaňková, Radka ; Beránek, Martin (advisor) ; Šimůnek, Tomáš (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Radka Vaňková Supervisor: doc. PharmDr. Martin Beránek, Ph.D. Title of diploma thesis: Influence of Carrier Molecule Addition on Plasma Free Circulating Nucleic Acid Extraction Outline: The aim of this thesis was to compare the yields of cell-free DNA (cfDNA) extracted from blood plasma specimens with the addition of commercially available carrier molecules. According to the real-time polymerase chain reaction (qPCR) results, we chose the most optimal carrier molecules for routine isolation of cfDNA from blood plasma samples. Methods: cfDNA was isolated from aliquots of a pooled blood plasma by spin column extraction method NucleoSpin Plasma XS (Macherey-Nagel). We used 7 different types of carrier molecules as RNA carrier (Qiagen), Glycogen (Invitrogen), Poly (A) Polyadenylic acid (Roche), Linear Acrylamide (Invitrogen), Yeast transfer RNA (Ambion), Salmon sperm DNA (Invitrogen), Herring sperm DNA (Promega). After the extraction, plasmatic DNA with the addition of carrier molecules was quantified by spectrophotometry, fluorimetry and qPCR. Results: The best results were achieved with the addition of polyadenylic acid with final concentration of 1.55 μg/ml. The addition of carrier RNA and...
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Free circulating nucleic acids in plasma
Totzauerová, Kateřina ; Beránek, Martin (advisor) ; Šimůnek, Tomáš (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Kateřina Totzauerová Supervisor: Doc. PharmDr. Martin Beránek, Ph.D. Title of diploma thesis: Free circulating nucleic acids in blood plasma Outline: The aim of this thesis was to compare various commercially available DNA extraction kits and choose the universally optimal method for routine isolation of free circulating DNA from blood plasma samples according to their optical characteristics, the qPCR results and yield of short fragments. Methods: Four commercially available methods of extraction have been chosen to compare: QIAmp DNA Blood Mini Kit, QIAmp DSP Virus Spin Kit (both Qiagen), NucleoSpin Plasma XS (Macherey-Nagel) and Agencourt Genefind v2 (Beckman Coulter). DNA was isolated from aliquots of a pooled blood plasma of healthy individuals. Plasmatic DNA was quantified after extraction by spectrophotometry, fluorimetry and quantitative polymerase chain reaction. Fragmentation analysis on selected samples by capillary electrophoresis was also realized. Results: The best yield and purity provided the method from Qiagen QIAmp DSP Virus Spin Kit. Average value of the concentration determined qPCR was 49.95 ± 23.57 ng/mL and it gave the highest values of the fluorescence of...
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