Spotting optimization for oligo microarrays on aldehyde-glass

被引:20
作者
Dawson, ED
Reppert, AE
Rowlen, KL
Kuck, LR
机构
[1] InDevR, LLC, Boulder, CO 80301 USA
[2] Univ Colorado, Dept Chem & Biochem, Boulder, CO 80309 USA
关键词
low-density microarrays; diagnostics; spotting solution; aldehyde substrates; reproducibility;
D O I
10.1016/j.ab.2005.03.029
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Low-density microarrays that utilize short oligos (< 100 nt) for capture are highly attractive for use in diagnostic applications. yet these experiments require strict quality control and meticulous reproducibility. However, a survey of current literature indicates vast inconsistencies in the spotting and processing procedures. In this Study, spotting and processing protocols were optimized for aldehyde-functionalized glass substrates. Figures of merit were developed for quantitative comparison of spot quality and reproducibility. Experimental variables examined included oligo concentration in the spotting buffer.. composition of the spotting buffer, postspotting "curing" conditions, and postspotting wash conditions. Optimized conditions included the use of 3-4 mu M oligo in a 3x standard saline citrate/0.05% sodium dodecyl sulfate/0.001% (3-[(3-cholamidopropyl) dimethylammonia]-1-propane sulfonate) spotting buffer, 24-h postspotting reaction at 100% relative humidity, and a four-step wash procedure. Evaluation of six types of aldehyde-functionalized glass substrates indicated that those manufactured by CEL Associates, Inc. yield the highest oligo coverage. (c) 2005 Elsevier Inc. All rights reserved.
引用
收藏
页码:352 / 360
页数:9
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