The overall objective of the instrumentation project is the prototype development of user-friendly detection/imaging hardware with informatics software to support high throughput mRNA analyses of lung tissue samples in a microarray microtiter-based format. The custom instrumentation to be developed for the Program Project is based on the proximal CCD detection/imaging technology established at Genometrix under both federal (DOC/ATP, NIH, NASA) and private (venture capital, corporate partners) funding. The functional specifications for the instrumentation developed for the Program Project will facilitate high sensitivity for chemiluminescent and fluorescent labeling, aray multiplexing, high throughput, automated quantitation, and information processing. Specifically, the instrumentation workstation will accommodate both the fluorescent and chemiluminescent labeling formats proposed in Project 1. Several motifs utilizing Eu/+3 chelators will be accommodated including linkage to beads in a sandwich assay, covalent attachment to mRNA, and simple sandwich assays using enzyme linked fluorescence. The proposed proximal CCD detection system will be automatically time gated to reduce background fluorescence. The proposed proximal CCD detection system will be automatically time gated to reduce background fluorescence, thereby taking full advantage of the long emission lifetimes characteristics of the chelating compounds. Also dioxitane chemiluminescent labeling will be employed wherein both linkage to detector probes and to beads in a sandwich assay will be accommodated. Finally, the informatics component of the workstation involves converting the pattern of array binding patterns into relative mRNA concentrations. Approaches to be investigated for this deconvolution problem include conventional statistical correlation analysis as well as neural networks and multicomponent maximum entropy methods. Following prototype development, the above instrumentation will be made available to scientists in Core A for use by all Program Project members. Also the workstations will be built in close collaboration with Program Project members, (especially members from Project 1, Core A and B) to ensure high compatibility with the multiparameter lung tissue mRNA analyses.
