Experimental analysis of hybridization chamber, printing tip, and slide chemistry designs on DNA microarray fabrication and performance

DNA microarrays are an important biological tool for high throughput gene expression and DNA analysis, but much microarray data is unusable because of poor repeatability, caused in part by variations in microarray fabrication and experimental techniques. We sought to characterize and identify the causes of variability in microarray slide attachment chemistries, contact printing, and hybridization that contribute to poor repeatability in microarray analysis.; We tested 9 different types of microarray slides that included aldehyde, amine, poly-L-lysine, and carboxylic acid slide chemistries. Hybridization capacity for all slide chemistries varied with printing concentration of probe DNA. Probe spots printed on aldehyde chemistry had approximately 10 times the hybridization capacity of amine based chemistries and the lowest background levels. This chemistry provided the highest dynamic range and greatest sensitivity to low levels of target DNA.; We tested printing variations of probe deposition, spot diameter, and intra-spot uniformity from a commonly used microarray quill pin under conditions of relative humidity (R.H.) ranging from 40% to 80%, and with hygroscopic additives 1.5M betaine, and 50% DMSO. Significant printing variation was observed at all ranges of relative humidity and with printing solution additives. Evaporation of printing solution from the pin appeared to be the primary cause of printing variation and affected both probe concentration and deposition characteristics of the pin. A novel capillary pin was designed that protected pin contents from evaporation and significantly reduced printing variation without increased relative humidity or printing solution additives.; We tested microarray hybridization using an array of identical probe spots to measure hybridization levels and hybridization uniformity for coverslip “static” microarray hybridization and a novel chaotic advection hybridization method. Four hour chaotic advection hybridization increased hybridization levels approximately 2 to 4 fold over 24 hour static hybridization levels. Chaotic advection also produced more uniform hybridization over the array.; Optimization of slide chemistry and improved consistency in microarray printing, and hybridization could improve the repeatability of microarray analysis, enabling more data to be used and increasing its quantitative capabilities.