Probably the most versatile assay for genomic scale research is the high-density microarray. Microarray analysis has the potential for uncovering the complex behavior of gene expression. Microarray trends are becoming established which includes higher throughput and a need to analyze pg quantities of mRNA. This proposal is focused on one aspect of microarray technology (aRNA amplification) which plans to meet the observed trends of the industry. The current multi-step amplification process introduces a source of variability, time and expense. As the amount of input mRNA is lowered, likewise, confidence is reduced and the array experiments themselves have questionable validity. We recognized this limitation as an opportunity that our research group should study, specifically, how can we use our expertise and our supply of high quality transcription reagents to establish a R&D program to enhance the performance of RNA amplification methods? We will design an exhaustive optimization plan to explore a large matrix of conditions, work that is most suitable for a robotic platform. Careful microarray analysis with numerous internal controls will be used to monitor the progress of the optimization program to insure that the representation is accompanied enhanced yields. The optimized enzymatic steps will be integrated on to automated platforms, which will increase the throughput and consistency of the method for research demanding a large number of samples. The effort spent creating the robotic program will serve two purposes. First, it will allow us to screen a large space of reaction conditions (finding optimal reactants) it also be instrumental for creating the next versions of RNA amplification kits, specifically for common robots. The goal of the larger study is to further optimize each step of the method for the commercialization of kits, robotic reagent packages and finally a microfluidic device and accompanying reagents.
