Method

Two-color, rolling-circle amplification on antibody microarrays for sensitive, multiplexed serum-protein measurements

Heping Zhou¹, Kerri Bouwman¹, Mark Schotanus¹, Cornelius Verweij², Jorge A Marrero³, Deborah Dillon⁴, Jose Costa⁴, Paul Lizardi⁴ and Brian B Haab¹

¹  The Van Andel Research Institute, 333 Bostwick, Grand Rapids, MI 49503, USA

²  The University of Amsterdam, Department of Molecular and Cell Biology, 1081 BT Amsterdam, The Netherlands

³  The University of Michigan Medical School, 1500 East Medical Center Drive, Ann Arbor, MI 48109, USA

⁴  Yale University School of Medicine, Department of Pathology, 310 Cedar Street, New Haven, CT 06510, USA

author email corresponding author email

Genome Biology 2004, 5:R28

Published:	30 March 2004

Subject areas: Genome studies, Molecular biology, Methods, Biochemistry and structural biology

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.