Serum and plasma sample preparation
Serum and plasma samples were obtained from three different sources. Numbers of samples as well as type of processing are also given in Table . Serum samples from the Austrian Institute of Technology (source 1), age and sex matched, mean age of 27, taken from healthy volunteers, were prepared as follows: incubation of blood in Vacuette 9 ml Z Serum Clot Activator (Greiner Bio One, Frickenhausen, Germany) for 30 min followed by centrifugation at 1,800 × g for 10 min at room temperature. Serum-aliquots of 2 ml were stored at -80°C. The blood plasma was taken from the same donors as the serum at a single withdrawal. For the isolation of plasma, the blood samples were taken with a BD Vacutainer (BD diagnostics) glass whole blood tube with K3EDTA, centrifuged at 1300 × g for 10 min at 4°C and stored in 500 μl aliquots at -80°C (source 1).
Overview of sample processing
Serum samples provided by the Austrian Red Cross Blood Center (source 2) were treated according to the protocol described above. These serum samples were arranged into three sex matched subgroups: group 1 (36-45 years old), group 2 (46-55 years old), group 3 (56-65 years old). These samples were qualified as blood donors according the eligibility criteria of the Austrian Red Cross and are denoted as control individuals.
Samples obtained from the General Hospital in Vienna (AKH) consisting of control individuals, breast cancer patients and patients with metastasizing breast cancer were centrifuged at 1000 × g for 15 min and stored in 200 μl aliquots at -80°C until usage (source 3). The non-cancer patients of the AKH were defined upon personal interviews and examinations to exclude any familial history of breast cancer, any papable breast-nodules and any history or presence of cancerous disease.
Both the Red Cross Blood Center and the AKH provided anonymized samples after completion of all testing procedures and according to the institute's guidelines. The study was approved by the ethics committee of the Medical University Vienna and was carried out in compliance with the Helsinki Declaration.
Protein expression and MBD-bead assembly
The E. coli strain BL21, containing the pET6HMBD plasmid, kindly provided by Sally H. Cross [24
], was grown in Luria-Bertani medium with 30 μg/ml chloramphenicol and 50 μg/ml ampicillin. LB medium (10 ml) was inoculated with glycerol stock of E.coli strain BL21. Bacteria were grown for 8 h at 37°C and then further cultured over night in 250 ml LB medium. Culture was then split into three main cultures of 750 ml LB medium each for further culturing. The main cultures were grown in 2 l flasks for approximately 8 h, until an optical density (600 nm) of 0.6 was reached. Then recombinant protein expression was induced by adding Isopropyl-ß-D-thiogalactopyranosid (IPTG) to a final concentration of 0.4 mM. After incubation at 37°C over night, the E. coli biomass was collected by centrifugation at 4,000 rpm for 20 min. The E. coli biomass (approximately 3 g/l LB medium) was washed twice with PBS and resuspended in lyses buffer (20 mM HEPES, 1 M NaCl, 2 M Urea, 10% glycerol, 0.5 mM EDTA, 0.1% TritonX, pH = 8) to reach a concentration of 0.3 g biomass per ml. Samples were stored at -20°C until usage.
For MBD protein purification E.coli cells (800 μl) were mixed with 500 μl lyses buffer and lysed by repeated bead whirling mixing for 30 s in Lyses Matrix A tubes (MP Biomedicals, Eschwege, Germany) on a FastPrep24 instrument (MP Biomedicals). The bacterial extract was then centrifuged at 13,000 rpm for 10 min and the supernatant transferred to a new vial. Centrifugation at 13,000 rpm for 10 min was repeated until all cell debris was spun down.
A volume of 200 μl of a 50% suspension of Ni-Sepharose beads (Adar Biotech, Rehovot, Israel) were washed in 500 μl water, followed by centrifugation for 1 min at 1000 × g and removal of the supernatant from the beads. The beads were then equilibrated by adding 500 μl buffer A (20 mM HEPES, 100 mM NaCl, 10% glycerol, 20 mM beta-Mercaptoethanol (added fresh daily), 0.5 mM PMSF, 0.1 mM TritonX, pH = 8) and centrifuged at 1000 × g for 1 min. This procedure was repeated twice and then the beads were resuspended in 100 μl buffer A. His-tagged MBD-protein was bound to the equilibrated beads by addition of 800 μl bacterial extract (derived from approximately 250 mg E.coli wet cell weight) and 500 μl 2 × buffer A, and incubated for two hours on ice at which the beads were held in suspension upon repeated mixing.
MBD-loaded beads (200 μl) were washed twice by adding 500 μl wash buffer (buffer A, plus 10 mM Imidazol) followed by centrifugation at 1,000 × g for 1 min. The MBD protein was either eluted from beads with buffer A plus 500 mM Imidazol or used for DNA isolation. Protein concentration was measured with a BioRad Dc-Protein assay (Bio-Rad laboratories). Protein purification was visualized on a SDS page gel (NuPage Novex Bis-Tris Gel, Invitrogen, Lofer, Austria) by applying aliquots of 6.5 μl from each purification step and a dilution of 1 μl crude lysat in 10 μl loading-volume onto the gel. Proteins were mixed with 2.5 μl loading dye (Bio-Rad laboratories) and 1.5 μl Reducing agent (BioRad laboratories), denatured via 10 minutes at 70°C and loaded onto the gel, where 200 V were applied for 35 minutes.
MBD loaded bead preparation for DNA purification was performed as follows: Residual E.coli DNA was removed with buffer B (1.5 M NaCl, 20 mM HEPES, 20 mM β-Mercaptoethanol, 0.5 mM PMSF, 0.1 mM TritonX, pH = 8). The beads (100 μl) were then resuspended in 100 μl buffer A.
MBD-loaded bead-based DNA Isolation
For DNA extraction using the MBD immobilized Ni-beads, 1 ml of serum was diluted with 1 ml of 2 × buffer A and incubated with an aliquot of 60 μl of prepared 50% MBD loaded bead suspension in buffer A for 2 h on a thermo mixer (Eppendorf, Hamburg, Germany) at 450 rpm at room temperature. After centrifugation at 1000 × g for 1 min beads were separated and used for DNA isolation. The supernatant was used for autoantibody-profiling (see below).
The MBD-loaded beads including the bound serum DNA were washed twice with buffer C (20 mM Hepes, 100 mM NaCl, 10% glycerol, pH = 8) and resuspended in 145 μl 10 mM Tris-Cl buffer, pH = 8. DNA was eluted from MBD loaded beads by ProteinaseK-digestion (20 mg/ml, Fermentas, St.Leon-Rot, Austria). The reaction contained 5 μl ProteinaseK in a volume of 150 μl and was incubated at 55°C for 20 min followed by an incubation at 65°C for 20 min. DNA was then isolated from the ProteinaseK reaction supernatant using Quiagen MinElute columns (Quiagen, Venlo, Netherlands). Purification was performed according to manufacturers instruction with an elution volume of 17 μl.
Silica membrane-based DNA Isolation
DNA isolation from serum using common silica membrane-based isolation strategy was performed applying the Roche High pure template preparation kit (Roche Diagnostics, Mannheim, Germany). The protocol was adapted according to Müller HM. et al. [25
]. Isolation was performed according to the manufacturer's instruction except the following protocol steps: the 800 μl of serum samples were split into 2 aliquots of 400 μl and each mixed with 400 μl of Roche Binding Buffer and 80 μl ProteinaseK (20 mg/μl Fermentas, St.Leon-Rot, Austria). After 15 min of incubation at 55°C, 200 μl isopropanol was added to each aliquot. Aliquots were mixed and an aliquot of 540 μl per serum sample were subsequently loaded four times to the column followed by a centrifugation step of 1 min at 8,000 × g. The flow through after each centrifugation was transferred back onto the same column and centrifuged again. Inhibitor-removal and washing steps were performed according to manufacturers' protocol, but the DNA was eluted in 55 μl of elution buffer.
DNA concentration was measured with the "Quant it" Pico Green kit (Invitrogen, Lofer, Austria) according to the manufacturers' protocol with a reduced reaction volume of 100 μl. Samples and standards were excited at 480 nm and emission was read at 520 nm using a BioRad IQ5 Real time PCR detection system. A five-point lambda DNA standard concentration curve ranging from 15 pg/μl to 250 pg/μl was measured in a volume of 100 μl.
Microarray-based autoantibody tests
cDNA clones (fetal brain expression library) found reactive with serum from control individuals and breast cancer patients were used for protein expression and microarray generation (data not shown). Protein chips were processed as described by Stempfer R. et al. [26
Serum samples, all originating from source 1, were either diluted 1 to 10 for untreated serum or 1 to 5 for samples after MBD-DNA isolation with PBST containing 3% nonfat dried bovine milk. Slides were blocked with DIGeasy Hyb (Roche Diagnostics, Mannheim, Germany) for 30 min, followed by two 5 min washing steps with PBST. A volume of 150 μl of diluted serum was applied onto each array and incubated for 2 h at room temperature. Slides were washed twice with PBST and detection of serum auto-antibodies was performed by incubation with Cy3 conjugated Affini Pure rabbit anti human IgA + IgG + IgM (Jackson Immuno Research) in a dilution of 1:25000 in PBST plus 3% nonfat dried bovine milk for 2 h followed by a 5 min washing step in PBST. Slides were scanned on a Gene Pix 4000a scanner (Axon Instruments) with a resolution of 10 μm at a photomultiplier tube setting of 700 PMT. Images were analyzed using Gene Pix software. Raw data were imported to GraphPad Prism (GraphPad Software, Inc.) to create Pearson correlation plots and to determine average signal intensities.
DNA amplification and qPCR tests
DNA recovery of distinct isolation methods was controlled by two Multiplex-PCRs, which were performed with 6 primer pairs per reaction targeting the 5'-UTRs of CpG methylation controlled genes (primer sequences on request). An aliquot of 2 μl per serum DNA isolate served as template for the reaction and the (2-step)-PCR reaction setup and cycling was performed as published previously [27
]. PCR products (10 μl) were loaded onto a 2% agarose gel containing 0.5 μg/ml ethidiumbromide.
qPCR analyses were carried out to assess the DNA integrity and enrichment of methylated DNA. All qPCR reactions were performed in a 384 well format on a Roche Light cycler 480 in reaction volumes of 10 μl containing 0.125 μM of each Primer, 0.3 U Hotstart Taq (Qiagen), 5% DMSO containing SYBR green to reach a dilution of 0.5 × in the final reaction and 166 μM dNTP-mix. A volume of 2 μl of Silica-membrane based DNA isolates served as template, whereas 1 μl template was used for the qPCR reaction of serum or plasma isolates, processed with MBD loaded beads. The PCR program was identical to the multiplex PCR program with the exception that for qPCR analyses 50 cycles were performed. Accuracy of Ct-values was assured via melting curve analyses of every analyzed reaction.
Mean and standard derivations were calculated for each data set. Pearson correlation coefficients were applied as a measurement of similarity between the data sets. A Wilcoxon rang sum test was applied for the not normally distributed data set of elevated DNA levels. None of the discussed data sets were normalized.