Bernd Schmidt1, Dana Reinicke2, Iris Reindl2, Ines Bork2, Bettina Wollschläger2, Nina Lambrecht2, Michael Fleischhacker3. 1. DRK Kliniken Berlin/Mitte, Klinik für Innere Medizin - Schwerpunkt Pneumologie und Schlafmedizin, Drontheimer Straße 39-40, 13359 Berlin, Germany. 2. Universitätsklinikum Halle/Saale, Klinik f. Innere Medizin, Abtlg. Pneumologie, Ernst Grube Str. 40, 06120 Halle/Saale, Germany. 3. Universitätsklinikum Halle/Saale, Klinik f. Innere Medizin, Abtlg. Pneumologie, Ernst Grube Str. 40, 06120 Halle/Saale, Germany. Electronic address: michael.fleischhacker@uk-halle.de.
Abstract
BACKGROUND: In most research laboratories the use of EDTA tubes for the isolation of plasma DNA from tumor patients is standard. Unfortunately these tubes do not allow for an extended storage of samples before processing and prevent EDTA tubes from being shipped at ambient temperature. The aim of our study was to compare the quantity and quality of plasma DNA isolated from EDTA and PAXgene® Blood ccfDNA Tubes in different downstream applications. METHODS: We enrolled 29 patients in our study. Blood samples were drawn into EDTA and PAXgene® Blood ccfDNA Tubes and were processed on day 0 and day 7 after storage at ambient temperature. The plasma DNA from 10 patients was isolated manually. For the DNA isolation from the plasma of 19 additional patients we used the automated QIAsymphony system. The total amount DNA from all samples was measured with a quantitative real-time PCR assay. In addition the amount of methylated mSHOX2 plasma DNA was determined. RESULTS: While the 7day storage lead to an increased amount of total DNA in almost all EDTA tubes, this effect was only seen in very few PAXgene® Blood ccfDNA Tubes. The stabilization solution which prevents the lysis of blood cells had no effect on the method for quantification of methylated sequences in these samples. CONCLUSION: The quantity and quality of plasma DNA from both types of blood draw tubes are comparable. DNA from PAXgene® Blood ccfDNA was successfully used for PCR-based quantification of total amount of cell-free DNA and for methylation analysis as well.
BACKGROUND: In most research laboratories the use of EDTA tubes for the isolation of plasma DNA from tumorpatients is standard. Unfortunately these tubes do not allow for an extended storage of samples before processing and prevent EDTA tubes from being shipped at ambient temperature. The aim of our study was to compare the quantity and quality of plasma DNA isolated from EDTA and PAXgene® Blood ccfDNA Tubes in different downstream applications. METHODS: We enrolled 29 patients in our study. Blood samples were drawn into EDTA and PAXgene® Blood ccfDNA Tubes and were processed on day 0 and day 7 after storage at ambient temperature. The plasma DNA from 10 patients was isolated manually. For the DNA isolation from the plasma of 19 additional patients we used the automated QIAsymphony system. The total amount DNA from all samples was measured with a quantitative real-time PCR assay. In addition the amount of methylated mSHOX2 plasma DNA was determined. RESULTS: While the 7day storage lead to an increased amount of total DNA in almost all EDTA tubes, this effect was only seen in very few PAXgene® Blood ccfDNA Tubes. The stabilization solution which prevents the lysis of blood cells had no effect on the method for quantification of methylated sequences in these samples. CONCLUSION: The quantity and quality of plasma DNA from both types of blood draw tubes are comparable. DNA from PAXgene® Blood ccfDNA was successfully used for PCR-based quantification of total amount of cell-free DNA and for methylation analysis as well.
Authors: Laure Sorber; Karen Zwaenepoel; Julie Jacobs; Koen De Winne; Kaat Van Casteren; Elien Augustus; Filip Lardon; Hans Prenen; Marc Peeters; Jan Van Meerbeeck; Geert Roeyen; Christian Rolfo; Patrick Pauwels Journal: Mol Diagn Ther Date: 2020-02 Impact factor: 4.074
Authors: Mark Southwood; Tomasz Krenz; Natasha Cant; Manisha Maurya; Jana Gazdova; Perry Maxwell; Claire McGready; Ellen Moseley; Susan Hughes; Peter Stewart; Manuel Salto-Tellez; Daniel Groelz; Doris Rassl Journal: J Pathol Clin Res Date: 2019-11-11
Authors: Zuzana Pös; Ondrej Pös; Jakub Styk; Angelika Mocova; Lucia Strieskova; Jaroslav Budis; Ludevit Kadasi; Jan Radvanszky; Tomas Szemes Journal: Int J Mol Sci Date: 2020-11-16 Impact factor: 5.923