Methyl-binding domain protein-based DNA isolation from human blood serum combines DNA analyses and serum-autoantibody testing
1 Molecular Medicine, Austrian Institute of Technology, Muthgasse 11, 1190 Vienna, Austria
2 Department of Obstetrics and Gynecology, Medical University of Vienna, Währinger Grütel 18 - 20, 1090 Vienna, Austria
3 Blood Donation Center for Vienna, Lower Austria and Burgenland, Austrian Red Cross, Wiedner Hauptstraße 32, 1040 Vienna, Austria
BMC Clinical Pathology 2011, 11:11 doi:10.1186/1472-6890-11-11Published: 6 September 2011
Additional file 1:
Differential methylated DNA fragments. Ct-values of differential methylated DNA fragments: DNA was isolated from serum (source 1, n = 8) either using the silica membrane based approach or the MBD approach. Four different 5'-UTR regions with known methylation status in healthy peripheral blood were tested, SALL3, ESR1, CHFR (all unmethylated loci) and ZNF502 (biallelic methylated loci). A mean difference between Ct-values regarding the three unmethylated genes of 2.2 was observed; highlighting the reduced amounts of unmethylated DNA, isolated by MBD loaded bead approach. For the ZNF502 gene locus, which is methylated in healthy adults, no significant difference between Ct-values of two isolation approaches was detected.
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Additional file 2:
SDS gel of MBD purification. The Coomassie stained SDS gel outlines our simplified MBD purification protocol and enabled us to control correct MBD-bead assembly. Aliquots of the several purification steps were loaded onto the gel. Lane 1, 5 kDa Page Ruler; Lane 2, crude lysate (1:5 dilution); Lane 3, supernatant of the binding reaction; Lane 4 and 5, subsequent washing steps with buffer A containing 10 mM imidazol; Lane 6, purified MBD protein eluted from Ni-NTA beads, where one band at 11 kDa remained.
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