In this master's thesis, we evaluated and compared different methods for DNA extraction and quantification from fresh-frozen and FFPE tumor samples, from brain tumor glioblastoma, with the aim of establishing a standardised approach to molecular diagnostics. DNA was extracted using the Qiagen AllPrep DNA/RNA/Protein Mini Kit, Qiagen QIAamp DNA FFPE Kit, PROMEGA Maxwell RSC Whole Blood DNA Kit and PROMEGA Maxwell RSC DNA FFPE Kit. The quantity of isolated DNA was assessed using a spectrophotometric method (NanoDrop), a fluorimetric method (Qubit), automated electrophoresis (TapeStation), and digital PCR as a reference method. The results demonstrated the differences in DNA yield among all extraction methods, variations in DNA concentration determined by different quantification approaches, and a strong influence of input material type. Fresh-frozen samples enabled the isolation of DNA with higher integrity and higher concentrations, whereas FFPE samples yielded lower amounts of DNA and showed increased levels of impurities. NanoDrop predominantly reported measurements with higher DNA concentrations, Qubit provided more consistent and, compared to NanoDrop, measurements with lower concentrations. TapeStation was mainly useful as a complementary method for DNA integrity determination. dPCR is the most robust approach for absolute DNA quantification. These findings confirm that there is no single universal method and higlight the importance of combining optimised extraction protocols with appropriate quantification methods to better support diagnostic analysis protocols in clinical practice.
|
