Zhou Heping, Bouwman Kerri, Schotanus Mark, Verweij Cornelius, Marrero Jorge A, Dillon Deborah, Costa Jose, Lizardi Paul, Haab Brian B
The Van Andel Research Institute, 333 Bostwick, Grand Rapids, MI 49503, USA.
Genome Biol. 2004;5(4):R28. doi: 10.1186/gb-2004-5-4-r28. Epub 2004 Mar 30.
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.
便捷快速地分析多种蛋白质的能力具有重要的应用价值。为进一步实现这一功能，我们开发了滚环扩增（RCA）技术，用于测量分别用生物素和地高辛标记的两种血清样本中蛋白质的相对水平，这些样本已被捕获在抗体微阵列上。与使用基于聚丙烯酰胺水凝胶和硝酸纤维素制备的抗体微阵列的直接标记和间接检测方法相比，双色RCA产生的荧光高出30倍。对24份血清样本中的多种蛋白质进行的重复RCA测量具有高度的可重复性和准确性。此外，RCA能够对低丰度蛋白质的不同表达谱进行可重复测量，而其他检测方法无法测量这些蛋白质。抗体微阵列上的双色RCA应该能够方便地获取各种蛋白质的表达谱，以用于各种应用。
