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Two-color,rolling-circle amplification on antibody microarrays for sensitive,multiplexed serum-protein measurements
Authors:Heping?Zhou  Kerri?Bouwman  Mark?Schotanus  Cornelius?Verweij  Jorge?A?Marrero  Deborah?Dillon  Jose?Costa  Paul?Lizardi  Email author" target="_blank">Brian?B?HaabEmail author
Institution:(1) The Van Andel Research Institute, 333 Bostwick, Grand Rapids, MI 49503, USA;(2) Department of Molecular and Cell Biology, The University of Amsterdam, 1081 BT Amsterdam, The Netherlands;(3) The University of Michigan Medical School, 1500 East Medical Center Drive, Ann Arbor, MI 48109, USA;(4) Department of Pathology, Yale University School of Medicine, 310 Cedar Street, New Haven, CT 06510, USA;
Abstract:The ability to conveniently and rapidly profile a diverse set of proteins has valuable applications. In a step toward further enabling such a capability, we developed the use of rolling-circle amplification (RCA) to measure the relative levels of proteins from two serum samples, labeled with biotin and digoxigenin, respectively, that have been captured on antibody microarrays. Two-color RCA produced fluorescence up to 30-fold higher than direct-labeling and indirect-detection methods using antibody microarrays prepared on both polyacrylamide-based hydrogels and nitrocellulose. Replicate RCA measurements of multiple proteins from sets of 24 serum samples were highly reproducible and accurate. In addition, RCA enabled reproducible measurements of distinct expression profiles from lower-abundance proteins that were not measurable using the other detection methods. Two-color RCA on antibody microarrays should allow the convenient acquisition of expression profiles from a great diversity of proteins for a variety of applications.
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