Non-fasting venous blood was collected into EDTA-coated tubes, spun, separated into plasma, aliquoted and snap-frozen at -80C until sample processing.
Label
Cy3
Label protocol
Following the manufacturer’s protocol and utilizing SOMAmer reagents immobilized on streptavidin beads, proteins from plasma samples were tagged with NHS-biotin reagent, captured as a SOMAmer reagent/protein complex, cleaved, denatured, eluted and hybridized to a custom Agilent DNA microarray. Additional details can be found at http://www.tandfonline.com/doi/abs/10.1586/erm.10.89 and Gold L et al. N Biotechnol. 2012 Jun 15;29(5):543-9.
Hybridization protocol
Following the manufacturer’s protocol and utilizing SOMAmer reagents immobilized on streptavidin beads, proteins from plasma samples were tagged with NHS-biotin reagent, captured as a SOMAmer reagent/protein complex, cleaved, denatured, eluted and hybridized to a custom Agilent DNA microarray. Additional details can be found at http://www.tandfonline.com/doi/abs/10.1586/erm.10.89 and Gold L et al. N Biotechnol. 2012 Jun 15;29(5):543-9.
Scan protocol
Microarrays were scanned with an Agilent SureScan scanner at 5 um resolution, and the Cy3 fluorescence readout was quantified. Additional information can be found in Gold L et al. N Biotechnol. 2012 Jun 15;29(5):543-9.
Data processing
Raw signal values were processed using Somalogic's SOMAscan standardization procedures, including hybridization normalization, plate scaling, median scaling, and final somamer calibration as described in http://www.tandfonline.com/doi/abs/10.1586/erm.10.89.
